Fusion proteins comprising an engineered knottin peptide and uses thereof

ABSTRACT

The present disclosure presents a general approach to engineering existing protein-protein interactions through domain addition and evolution. The disclosure teaches the creation of novel fusion proteins that include knottin peptides where a portion of the knottin peptide is replaced with a sequence that has been created for binding to a particular target. Such fusion proteins can also be bispecific or multi specific in that they can bind to and/or inhibit two or more receptors or receptor ligands. Knottins may be fused with an existing ligand (or receptor) as a general platform for increasing the affinity of a ligand-receptor interaction or for creating a multi specific protein. In addition, the fusion proteins may comprise a knottin peptide fused to another protein where the other protein facilitates proper expression and folding of the knottin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional PatentApplication No. 61/411,350 filed on Nov. 8, 2010, which is herebyincorporated by reference in its entirety.

STATEMENT OF GOVERNMENTAL SUPPORT

This invention was made with Government support under contract CA151706awarded by the National Institutes of Health. The Government has certainrights in this invention.

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. The sequence listing was created Nov. 7,2011, has 61,440 bytes and is named “381593 pct.txt”.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the field of protein engineering, andto the field of knottin peptides, i.e. peptides with particularlywell-defined scaffolds and high stability, also referred to as cystineknot miniproteins in the art.

2. Related Art

Presented below is background information on certain aspects of thepresent invention as they may relate to technical features referred toin the detailed description, but not necessarily described in detail.That is, individual parts or methods used in the present invention maybe described in greater detail in the materials discussed below, whichmaterials may provide further guidance to those skilled in the art formaking or using certain aspects of the present invention as claimed. Thediscussion below should not be construed as an admission as to therelevance of the information to any claims herein or the prior arteffect of the material described.

Protein-protein interactions mediate nearly every process in livingsystems and gene duplication and recombination is believed to becritical to the evolution of protein function. Directed evolution is aninvaluable tool for optimizing proteins, however, in vitro evolutionstrategies generally focus on directly engineering the active site orbinding site of the protein of interest. There are limited examplesharnessing the power of gene duplication and combination in the directedevolution of protein function.

Specific molecular recognition events define the interactions betweenligands and receptors in living systems. These interactions mediate ahost of biological processes, highlighting the importance of molecularrecognition in many physiological processes. Engineering molecularrecognition has been widely used in the biotechnology arena to developprotein-based biosensors, imaging agents, and therapeutics candidates.Traditional approaches for engineering enhanced recognition focus onoptimizing the specific interaction, for example enhancing antibodyrecognition or affinity maturation of native protein-proteininteractions. In nature, however, molecular recognition often occurs atthe interface of multiple domains, and the linkage of protein domainsthrough gene recombination is believed to play a strong role in theevolution of protein function. There are few instances in the literatureof this approach being used to engineer protein function in vitro.Examples that do exist are limited to either evolving a completelysynthetic interaction or optimizing a protein-peptide interaction. Inthe same way that traditional directed evolution studies have providedinsights into the natural evolution of proteins, harnessing nature'sapproach of domain addition and evolution would provide new avenues toexplore natural evolution pathways. Further analysis of domain additionand evolution, focusing on enhancing an existing high affinityprotein-protein interaction, would provide a rigorous test of theutility of this approach for the study of molecular recognition and foruse as a protein engineering tool.

SPECIFIC PATENTS AND PUBLICATIONS

Knottins are described in the knottin database, http(colon slash slash)knottin.cbs.cnrs.fr/Knottins.php, which provides sequences andstructures of various knottin peptides.

U.S. Pat. No. 7,674,881 to Kent, et al., issued Mar. 9, 2010, entitled“Convergent synthesis of proteins by kinetically controlled ligation,”describes the synthesis of EETI-II.

Liu U.S. Pat. No. 5,468,634, entitled “Axl oncogene”, discloses isolatedDNA sequences encoding a mammalian axl receptor which exhibits axloncogene activity.

US 2009/0257952 to Cochran et al., published Oct. 15, 2009, entitled“Engineered Integrin Binding Peptides,” discloses engineered peptidesthat bind with high affinity (low equilibrium dissociation constant(K_(D))) to the cell surface receptors of fibronectin (alpha 5 beta1integrin) or vitronectin (alpha v beta 3 and alpha v beta 5 integrins).

BRIEF SUMMARY OF THE INVENTION

The following brief summary is not intended to include all features andaspects of the present invention, nor does it imply that the inventionmust include all features and aspects discussed in this summary. For thesake of brevity, it is to be understood that certain features ofdifferent embodiments may be combined, even though such alternativecombinations or subcombinations are not explicitly recited.

Thus, in certain aspects, the present invention comprises (a) a knottinpolypeptide having therein a binding loop for binding to a first target;and (b) a second polypeptide having therein a sequence for binding to asecond target, said second polypeptide being either (i) a cell surfacereceptor binding to said second target or (ii) a cell surface receptorligand. binding to said second target. As is known in knottins, bindingloops are typically between constrained cysteine residues. These loopsmay be altered by preparing a library of randomized sequences. In thisaspect, the knottin polypeptide contains a non-native sequence in itsbinding loop. That is, the sequence is not normally present in theknottin; preferably it has been selected by a screening procedure forhigh binding. In certain aspects of the invention, the fusion proteinwill contain a non-native sequence mediates attachment between a celland the tissues surrounding it. In certain aspects of the invention, theknottin polypeptide contains a sequence that mediates binding to one ormore of (a) alpha v beta 3 integrin, (b) and alpha v beta 5 integrin,and (c) alpha 5 beta 1 integrin. In certain aspects of the invention,the fusion protein comprises a second polypeptide which is anextracellular domain of a receptor tyrosine kinase. In certain aspectsof the invention, the second polypeptide is a receptor tyrosine kinaseIg1 domain. In certain aspects of the invention, the Ig1 domain is fromAxl, MuSK, or the FGF receptor. In certain aspects of the invention, thereceptor tyrosine kinase is an Axl receptor. In certain aspects of theinvention, the knottin polypeptide is selected from the group consistingof EETI-II, AgRP, and agatoxin. In certain aspects of the invention, thefusion protein has a binding loop domain is engineered to bind to one ofα5β1 integrin, αvβ3 integrin, or αvβ5 integrin.

In certain aspects of the invention, the fusion protein comprises (a) anEETI-II or AgRP knottin polypeptide comprising a binding loop with highaffinity to an integrin; and (b) a polypeptide selected from the groupconsisting of (i) an Axl extracellular domain and (ii) NK1 fragment ofhepatocyte growth factor.

Certain aspects of the invention comprise a method for preparing afusion protein, comprising the steps of: (a) preparing a library havinga number of DNA constructs encoding the fusion protein and a number ofrandomized DNA sequences within the DNA constructs; (b) expressing theDNA constructs in the library in yeast, wherein expressed DNA constructsare displayed as polypeptides with randomized sequences on the yeastsurface; (c) screening the clones for binding of the expressed DNAconstructs to the first target or the second target by contacting theclones with a target; (d) selecting clones that express translated DNAconstructs that bind with high affinity to the target; and (e) obtainingthe coding sequences of the selected clones, whereby said fusion proteinmay be prepared.

Certain aspects of the invention comprise a method for inhibitingbinding of a ligand to a receptor, comprising the steps of: (a)administering an amount of a soluble fusion protein comprising (i) apolypeptide encoding an extracellular domain of a receptor to beinhibited and (ii) a knottin polypeptide having a loop domain engineeredto bind to a cell surface receptor that is not the receptor to beinhibited.

In certain aspects of the various methods, the tyrosine kinase may be aTAM receptor tyrosine kinase.

In certain aspects, the present invention comprises a method forpreparing a bispecific, or multispecific, fusion protein that containsan engineered knottin portion and another binding portion that,preferably, is a receptor, receptor ligand, or a fragment thereof havingthe binding property of the native molecule. The fusion protein thusprepared has two different binding portions, and two separate ligands.The knottin portion is fused at its C-terminus to the N terminus of thebinding portion. Alternatively, it may be fused at its N terminus to theC terminus of the binding portion.

In certain aspects, the present invention comprises a method forpreparing a fusion protein comprising a first polypeptide that binds toa first binding partner (e.g. a receptor or receptor ligand) fused to asecond polypeptide (e.g. a knottin) having a loop domain engineered tobind with high affinity to a second binding partner, comprising thesteps of: (a) preparing a library having a number of DNA constructsencoding the fusion protein and a number of randomized loop domains,wherein the library provides a degree of variation of binding and anumber of tight binders to be selected from the library; (b) expressingthe DNA constructs in the library as protein variants; (c) screening thelibrary for binding of the protein variants to the second bindingpartner; (d) selecting clones that express DNA constructs that bind withhigh affinity to the second binding partner; and (e) obtaining thecoding sequences of the selected clones, whereby said fusion protein maybe prepared. The second binding partner selected may be an entirelydifferent molecule (protein, glycoprotein, polysaccharaide, lipid, cellstructure, viral epitope etc.) or it may be a different epitope on thebinding site for the first binding partner (receptor or receptorligand). In certain aspects, the present invention utilizes a firstpolypeptide that is a receptor fragment. For example, a cell surfacereceptor having various domains is used in the form of a fragmentencoding an extracellular ligand binding domain. The cell surfacereceptor may be a receptor tyrosine kinase. In certain aspects of theinvention, the first polypeptide may be a receptor ligand, or a fragmentof such a ligand that binds to a receptor. The ligand may be an agonistor an antagonist. The first polypeptide may have a sequence which is atleast a portion of a sequence selected from the group consisting of Axl,c-Met, HGF, VEGF, VEGF receptor, and Gas6.

In certain aspects of the present invention, the second polypeptide is aknottin scaffold and may be selected from the group consisting ofEETI-II, AgRP, and agatoxin. It is also contemplated that the knottinscaffold may be ω-conotoxin. In certain aspects of the presentinvention, the knottin loop domain is engineered to bind to an integrin.In certain aspects of the present invention, the method comprisescloning a random yeast display library having loop portions that areselected for binding to the target of interest.

In certain aspects, the present invention comprises a fusion proteincomprising a receptor ligand polypeptide, said receptor ligand bindingto a receptor at a specific receptor binding site, fused to a knottinpolypeptide having a loop domain engineered to bind with high affinityto a binding partner that is not the specific receptor binding site forthe receptor ligand. In certain aspects of the present invention, thereceptor ligand polypeptide is a fragment of a native ligand. In certainaspects of the present invention, the fusion protein comprises afragment that is a fragment of a growth factor, such as an NK1 fragmentof hepatocyte growth factor, which consists of the HGF amino terminusthrough the first kringle domain.

Certain aspects of the present invention comprise a fusion proteincomprising a receptor polypeptide, said receptor binding to a ligand ata specific ligand binding site, fused to a knottin polypeptide having aloop domain engineered to bind with high affinity to a binding partnerthat is not the specific ligand binding site. The receptor may be is areceptor tyrosine kinase. The receptor tyrosine kinase may be selectedfrom the group consisting of Axl, a receptor tyrosine kinase involved insolid tumor progression and MET, which is the hepatocyte growth factorreceptor. It may include closely receptor tyrosine kinases closelyrelated to Axl, such as Tyro-3 and Mer.

In certain aspects of the present invention the fusion protein comprisesa knottin polypeptide selected from the group consisting of EETI-II,AgRP, and agatoxin. In certain aspects of the present invention, thefusion protein comprises a loop domain engineered to bind to one of α₅β₁integrin, α_(v)β₃ integrin, or α_(v)β₅ integrin. In certain aspects ofthe present invention, the loop domain is engineered to bind to a β₃integrin. In certain aspects of the present invention, the loop domainis engineered to bind to an α_(v) or β₃ integrin subunit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic drawing of the Axl extracellular domain.

FIG. 1B is a ribbon rendering of an EETI-II crystal structure.

FIG. 1C is a schematic drawing of the Axl-EETI-II fusion bound to theGas6 ligand.

FIG. 1D is a representation of the EETI-II-axl fusion library creationand the screening to obtain fusions EA 7.01, 7.03, 7.05, 8.04 and 8.05.Both loops 1 and 2 can be seen to be randomized; only a portion of theAxl1 Ig1 sequence is represented. The sequences are truncated due to thelength of the Axl Ig1 portion.

FIG. 2A is a schematic drawing of the yeast display construct.

FIG. 2B is a set of scatter plots showing comparison of binding bywild-type Axl Ig1 and the starting E-Axl library

FIG. 3 is a set of scatter plots of results of EA-Axl library screeningand sort progression.

FIG. 4 is a graph that shows equilibrium binding of wild-type Axl Ig1,wild-type EETI-Axl, and EA (“EETI-II-Axl”) mutants to Gas6.Representative data of experiments performed in triplicate on separatedays.

FIG. 5 is a graph that shows kinetic dissociation of wild-type Axl Ig1or EA mutants from soluble Gas6. Wild-type Axl Ig1 was well fit by asingle exponential decay model, while EA mutants had to be fit with adouble-exponential decay model. Representative data of experimentsperformed in triplicate on separate days.

FIGS. 6A, 6B and 6C is a series of graphs that shows the contribution ofindividual loops in EA mutants. Reversion to wild-type for (6A) EA 7.01,(6B) EA 7.06, (6C) EA 8.04. wtL1 or wtL3 refers to wild-type EETI-IIloop sequence for loop 1 or loop 3, respectively. Persistent binding forwtEETI-Axl is shown on each plot for reference and represents“reversion” of both loops 1 and loop 3 to wild-type EETI-II sequence.Data is average of experiments performed on three separate days, errorbars are ±std. dev.

FIGS. 7A and 7B is a pair of bar graphs that shows the binding ofsurface displayed AgRP-Aras4 fusion protein against soluble α_(v)β₃integrin and Met protein compared with AgRP7A and NK1 mutant Aras4.

FIG. 8 is a line graph that shows binding titrations of the fusionprotein, AgRP7A-Aras4 to cells that express α_(v)β₃ integrin and Metreceptor.

FIGS. 9A and 9B are a pair of graphs showing binding to Gas 6 (9A) andalpha v beta 3 integrin (9B) of a Axl-EETI direct fusion protein.

FIG. 10 is a graph that shows the inhibition of PC3 tumor cell adhesionto microtiter plates coated with vitronectin. Knottin 2.5F-Fc and2.5D-Fc (knottin-integrin fused to Fc portions) inhibit PC3 celladhesion with concentrations in the low nanomolar range. Negativecontrol is an irrelevant protein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Overview

The present invention comprises the creation of novel fusion proteinsthat include an engineered knottin peptide fused to a second, differentpeptide or protein which provides a different binding function. Thesecond polypeptide is a receptor or a receptor ligand. Preferably, aportion of the knottin peptide is replaced with a sequence that has beencreated for binding to an integrin. In addition, the fusion proteins maycomprise a knottin peptide fused to another protein where the otherprotein facilitates proper expression and folding of the knottin.

The present invention may be used to enhance receptor ligand binding.Native proteins involved in ligand-receptor interactions are promisingstarting points for engineering therapeutic candidates. Traditionalapproaches to engineering protein-protein interactions have focused onoptimizing an existing interaction. In nature, however, protein-proteininteractions often occur at the junction of multiple domains and generecombination plays a strong role in the evolution of protein function.Using these observations, we have developed a general approach toengineering existing protein-protein interactions we refer to as “domainaddition and evolution” in which enhancement is accomplished byexpanding the binding interface through the addition and subsequent invitro evolution of a synthetic binding domain.

FIG. 1 shows that the present fusions in effect add another epitope forreceptor-ligand binding. FIG. 1A shows that the Axl extracellular domaincontains two immunoglobulin-like domains (Ig1 and Ig2), followed by twofibronectin type-III like (Fn) domains. FIG. 1B shows EETI-II crystalstructure (PDB ID: 2ETI). Loops 1 and 3, which were randomized fordomain addition and evolution library, are shown in black. CysteinesI-VI are noted. FIG. 1C is a schematic showing domain addition strategy.EETI-II mutant library is linked to the N-terminus of Axl Ig1 (blackribbons to the bottom left of the structure) to screen for EETI-IImutants that bind to an adjacent epitope on the Gas6 ligand. Axl-Gas6structure adapted from PDB ID: 2C5D. FIG. 1D shows a listing of aminoacid sequences that show the EETI-II loop 1 and loop 3 regions that wererandomized and the fusion to the Axl Ig1 domain. Figure was generatedusing PyMol.

FIGS. 2A and 2B shows the yeast display construct and evaluation ofstarting E-Axl library EETI-II mutants (randomized loops) linked to Axl.(2A) Yeast-displayed E-Axl construct. The protein of interest isexpressed as a genetic fusion to the yeast Aga2 protein, which isdisulfide bonded to the yeast Aga1 protein. The Aga1 protein iscovalently linked to the yeast cell wall, thereby tethering the entiredisplay construct to the yeast cell surface. The use of Aga1 and Aga2proteins in yeast display has been previously described in connectionwith surface display of antibodies. See, e.g. U.S. Pat. No. 6,423,538entitled “Yeast cell surface display of proteins and uses thereof,” byK. Dane Wittrup et al.

The HA and c-myc epitope tags flanking the protein of interest can bestained for relative yeast surface expression levels using commerciallyavailable antibodies (c-myc staining shown for reference). Soluble Gas6can be used to test binding to the yeast-displayed protein; Gas6 bindingis illuminated with a fluorescently labeled antibody against thehexahistidine tag (SEQ ID NO: 77) on Gas6. FIG. 2B presents scatterplots showing comparison of binding by wild-type Axl Ig1 and thestarting E-Axl library.

I. Knottin Fusions Having Bispecific or Multispecific Binding

In certain aspects, the present invention comprises fusion proteins thatare bispecific or multispecific in that they can bind to and/or inhibittwo or more receptors or receptor ligands for increased therapeuticefficacy. These fusions may comprise N-terminal or C-terminal knottinsengineered to contain, as one example, an integrin-binding portion.Integrin binding knottins are described in US 2009/0257952 by Cochran etal. entitled “Engineered Integrin Binding Peptides.” Engineered peptidesthat bind with high affinity (low equilibrium dissociation constant(K_(D))) to the cell surface receptors of fibronectin (α₅β₁ integrin) orvitronectin (α_(v)β₃ and α_(v)β₅ integrins) are disclosed. Knottins withnovel binding properties may be fused to generate hetero-oligomericbispecific proteins. This application is incorporated herein byreference, as provided in the concluding paragraph hereof, and may beconsulted further for descriptions of integrin-binding knottins. Thespecific integrin binding partner used here may be specific as to bothalpha and beta integrin chains, or only to a beta chain. In the lattercase, the integrin binding will be multispecific in that differentalpha-beta integrin combinations will exist.

For example, an integrin-binding knottin-ligand fusion has been createdusing a fragment of a growth factor, NK1. The integrin binding knottincontains a loop that has been engineered to bind specifically to aselected integrin, such as α₅β₁, α_(v)β₃, and α_(v)β₅, particularlyα_(v)β₃ integrins. NK1 is a fragment of the polypeptide growth factorHGF/SF which acts as agonist of the MET receptor. It is described morefully in US 2004/0236073 A1 by Gherardi, entitled “Nk1 fragment ofhepatocyte growth factor/scatter factor (hgf/sf) and variants thereof,and their use.” Briefly, HGF/SF has a unique domain structure thatresembles that of the blood proteinase precursor plasminogen andconsists of six domains: an N-terminal (N) domain, homologous toplasminogen activation peptide, four copies of the kringle (K) domainand a catalytically inactive serine proteinase domain. Two products ofalternative splicing of the primary HGF/SF transcript encode NK1, afragment containing the N and the first K domain, K1, and NK2, afragment containing the N, K1 and second kringle, K2, domains. Thesequence may be found in Mol Cell Biol, March 1998, p. 1275-1283, Vol.18, No. 3.

As another example, an integrin binding knottin-receptor fusion wasprepared using Axl. The Axl receptor is described in U.S. Pat. No.5,468,634 to Liu. Briefly, Axl is a receptor tyrosine kinase with astructure of the extracellular region that juxtaposes IgL and FNIIIrepeats. It is involved in the stimulation of cell proliferation. It canbind to the vitamin K-dependent protein Gas6, thereby transducingsignals into the cytoplasm. The extracellular domain of Axl can becleaved and a soluble extracellular domain of 65 kDa can be released.Cleavage enhances receptor turnover, and generates a partially activatedkinase (O'Bryan J P, Fridell Y W, Koski R, Varnum B, Liu E T. (1995) JBiol Chem. 270(2):551-557). However, the function of the cleaved domainis unknown.

The Axl receptor has two Gas6 binding sites (FIG. 1A): a major, highaffinity site located in its Ig1 domain, and a weaker minor site in itsIg2 domain. An active 2:2 signaling complex is formed when Gas6associates with Axl via its high affinity site, after which associationthrough the weak binding site results in receptor dimerization andactivation. This is a therapeutically relevant ligand-receptor system asAxl overexpression results in invasion and metastasis in a range ofcancer cell lines and inhibition of Axl signaling suppresses tumor cellmigration and metastasis. The bispecific protein generated binds withhigh affinity to integrins and the Axl ligand Gas6. FIG. 1 shows thatthe sequences represent an outline of domain addition and evolutionlibrary generation and screening; first row shows the wild-type EETI-IIsequence with cysteine bonds and loops between cysteines; second rowshows loops 1 and 3 where x residues are added; loops 1 and 3 of EETI-IIare randomized to generate the loop library and fused to the N-terminusof Axl Ig1; third row shows sequences of EETI-II-axl fusion mutants EA7.01, EA 7.06, and EA 8.04; bottom row lists sequences fromidentification of a PGM, or P-G/T-M/K motif.

The Axl amino acid sequence may be found in NCBI UniGene 26362, andGenbank Accession Number P30530.

In another aspect of the present invention, the receptor or other fusionprotein fused to the knottin, is also modified and mutated for bindingpurposes, in addition to being fused to a knottin that is mutated forbinding purposes. This is shown in Example 6. In this embodiment, thereceptor, which is to be used as a decoy, is first truncated to anextracellular domain. In the case of Axl, a portion of the signalpeptide and a small portion of the extracellular domain (about 110 aminoacids from the extracellular domain of about 426 amino acids were used).Using error-prone DNA amplification, mutations are introduced into theDNA sequence encoding the receptor fragment. The resulting clones arescreened for binding to the native ligand (Gas6 in the case of Axl), andtighter binders are selected, e.g. by cell sorting. A variety ofreceptor constructs could be used.

This knottin-Axl fusion can function as a bispecific or multispecificmolecule capable of concurrently antagonizing both integrin binding aswell as the native Gas6/Axl interactions. Gas6 is a soluble ligandwhereas the integrins are cell surface receptors, allowing both targetsto be bound at the same time. Binding of Gas6 will sequester the solubleligand, preventing it from associating with, and subsequently activatingendogenous Axl receptor. Binding to integrin receptors will prevent themfrom binding to extracellular matrix proteins.

The fusion of an integrin-binding peptide to a growth receptor or asignal transducing receptor such as a receptor tyrosine kinase isadvantageous in that there is significant cross-talk between integrinand growth factor receptor pathways. For example, strong cross-talkexists between integrins and Met receptor. An agent that targets bothreceptors will be better at inhibiting angiogenesis and metastasis.Integrin targeting by means of a fusion of a therapeutic protein and anintegrin-binding knottin can also localize the second therapeutic agentto the tumor cells, increasing efficacy through avidity effects.Moreover, an imaging agent that can target two tumor receptors wouldgenerate an increased signal and can detect smaller tumors for earlierdetection.

Knottin-Fc Fusions

Another example (see Example 12) of a fusion protein as described hereinis a fusion between an integrin binding knottin and an Fc portion of amouse antibody. The Fc portion of an antibody is formed by the twocarboxy terminal domains of the two heavy chains that make up animmunoglobin molecule. The IgG molecule contains 2 heavy chains (˜50 kDaeach) and 2 light chains (˜25 kDa each). The general structure of allantibodies is very similar, a small region at the tip of the protein isextremely variable, allowing millions of antibodies with slightlydifferent tip structures to exist. This region is known as thehypervariable region (Fab). The other fragment contains noantigen-binding activity but was originally observed to crystallizereadily, and for this reason was named the Fc fragment, for Fragmentcrystallizable. This fragment corresponds to the paired CH₂ and CH₃domains and is the part of the antibody molecule that interacts witheffector molecules and cells. The functional differences betweenheavy-chain isotypes lie mainly in the Fc fragment. The hinge regionthat links the Fc and Fab portions of the antibody molecule is inreality a flexible tether, allowing independent movement of the two Fabarms, rather than a rigid hinge. This has been demonstrated by electronmicroscopy of antibodies bound to haptens. Thus the present fusionproteins can be made to contain two knottin peptides, one on each arm ofthe antibody fragment.

The Fc portion varies between antibody classes (and subclasses) but isidentical within that class. The C-terminal end of the heavy chains formthe Fc region. The Fc region plays an important role as a receptorbinding portion. The Fc portion of antibodies will bind to Fc receptorsin two different ways. For example, after IgG and IgM bind to a pathogenby their Fab portion their Fc portions can bind to receptors onphagocytic cells (like macrophages) inducing phagocytosis.

The present knottin-Fc fusions can be implemented such that the Fcportion is used to provide dual binding capability, and/or for half-lifeextension, for improving expression levels, etc.

II. Knottin Fusions Used to Improve Ligand Receptor Binding

In this aspect of the present invention, a library of knottins having arandomized loop and fused to a receptor is screened and used as aplatform to create improved ligand binding. As one example, an EETIlibrary was fused to Axl, and this library was screened to isolateEETI-Axl binders with increased affinity to Gas6 ligand. Thus, knottinsmay be fused with an existing ligand (or receptor) as a general platformfor increasing the affinity of a ligand-receptor interaction.

Here we show the potential for the engineering of proteins through theaddition and subsequent optimization of a synthetic knottin bindingdomain. To demonstrate the power of this approach, we enhance a nativehigh affinity (single-digit nanomolar) protein-protein interaction tosubnanomolar levels using a single round of directed evolution. Throughthis work we also demonstrate that two structurally adjacent loops onthe surface of the Ecballium elaterium trypsin inhibitor II (EETI-II)knottin can be simultaneously engineered to form a binding face towardsan exogenous target. That is, a receptor and ligand may bind or be madeto bind at an additional surface by engineering of a loop on a fusedknottin, and/or engineering a loop in the receptor or ligand itself.This work demonstrates the potential for harnessing the naturalevolutionary process of gene duplication and combination for laboratoryevolution studies and should be broadly applicable to the study andoptimization of protein function.

The domain addition and evolution strategy is a broad-based strategy forenhancing affinity of existing protein-protein interactions. A syntheticbinding domain can be fused to the N- or C-terminus of a binding proteinand subsequently evolved to enhance affinity to the binding partner bybinding to an adjacent epitope. We also envision application inidentification of binding proteins from “naïve” libraries. By “naïve” wemean libraries based off of proteins with no native binding affinitytowards the target, e.g. the EETI-II knottin exhibits no native bindingaffinity towards Gas6. An additional application of this approachincludes identification of binding proteins from naïve libraries.EETI-II peptides engineered for binding tumor targets hold significantpromise for in vivo molecular imaging applications. However,identification of binding proteins from naïve libraries is challenging,in part due to the requirement that the affinity of the identifiedprotein must be high enough for detection. For example, in yeast surfacedisplay binding affinities in the single-digit μM range are below thelimits of detection and such proteins will generally not be enrichedduring library sorting. Domain addition and evolution can be used as an“anchoring” strategy, enabling identification of synthetic bindingdomains that enhance an existing interaction, but in isolation maythemselves possess affinity below the limits of detection. In theexample below, the EETI-II mutants developed here exhibit weak bindingaffinity towards Gas6 that are below the limits of detection when theknottin mutants are expressed in the absence of Axl. Subsequent affinitymaturation through traditional strategies or further domain addition andevolution can be used to generate fully synthetic binding agents withhigh affinity.

III. Knottin Fusions to Enhance Expression of Folded, Functional KnottinProteins

Knottin peptides may be difficult to obtain in properly folded form.Chemical synthesis and refolding of peptides may be done, but requiresextensive optimization. This problem can be mitigated by fusing theknottin to a protein. For example, EETI-II 2.5D (described below) couldnot be solubly expressed in yeast. However, when fused to Axl, a highyield of folded, functional knottin—Axl fusion was obtained. A proteasecleavage site was introduced between EETI-II 2.5D and Axl to cut off thefusion partner. This is a general strategy where any fusion partner canbe used for the expression, or it can be part of making a bispecificprotein as described above.

This will also have implications for fusing modifying domains, such asFc, human serum albumin, etc. to increase half-life for therapeuticapplications.

By fusing a difficult to express knottin to a well-expressed protein,yields can be improved. A protease recognition sequence is insertedbetween the knottin and the fusion partner. This is exemplified below inExample 7.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are described. Generally, nomenclatures utilized inconnection with, and techniques of, cell and molecular biology andchemistry are those well known and commonly used in the art. Certainexperimental techniques, not specifically defined, are generallyperformed according to conventional methods well known in the art and asdescribed in various general and more specific references that are citedand discussed throughout the present specification. For purposes ofclarity, the following terms are defined below.

The term “effective amount” means an amount of a fusion protein of thepresent invention that is capable of modulating binding of an engineeredpeptide to a cognate binding partner. The effective amount will dependon the route of administration and the condition of the patient.

“Pharmaceutically acceptable” is meant to encompass any carrier, whichdoes not interfere with the effectiveness of the biological activity ofthe active ingredient and that is not toxic to the host to which isadministered. For example, for parenteral administration, the aboveactive ingredients may be formulated in unit dosage form for injectionin vehicles such as saline, dextrose solution, serum albumin andRinger's solution.

The term “knottin protein” means a structural family of small proteins,typically 25-40 amino acids, which bind to a range of molecular targetslike proteins, sugars and lipids. Their three-dimensional structure isessentially defined by a peculiar arrangement of three to five disulfidebonds. A characteristic knotted topology with one disulfide bridgecrossing the macro-cycle limited by the two other intra-chain disulfidebonds, which was found in several different microproteins with the samecysteine network, lent its name to this class of biomolecules. Althoughtheir secondary structure content is generally low, the knottins share asmall triple-stranded antiparallel β-sheet, which is stabilized by thedisulfide bond framework. Biochemically well-defined members of theknottin family, also called cysteine knot proteins, include the trypsininhibitor EETI-II from Ecballium elaterium seeds, the neuronal N-typeCa2+ channel blocker ω-conotoxin from the venom of the predatory conesnail Conus geographus, agouti-related protein (AgRP, See Millhauser etal., “Loops and Links: Structural Insights into the Remarkable Functionof the Agouti-Related Protein,” Ann. N.Y. Acad. Sci., Jun. 1, 2003;994(1): 27-35), the omega agatoxin family, etc. A suitable agatoxinsequence is given in US 2009/0257952, having a common inventor with thepresent application. Another agatoxin sequence is given at GenBank®Accession number P37045, Omega-agatoxin-Aa4b; P81744,Omega-agatoxin-Aa3b, etc. Other knottin sequences may be found atGenBank® Accession number FJ601218.1, knottin [Bemisia tabaci]; GenbankAccession number P85079, Omega-lycotoxin; and Genbank Accessioin numberAAB34917, mu-O conotoxin MrVIA=voltage-gated sodium channel blocker.

Conotxins generally consist of peptides which are 10-30 residues inlength. A specific example is PRIALT® ziconotide, a synthetic equivalentof a naturally occurring conopeptide found in the piscivorous marinesnail, Conus magus. Ziconotide, which is a 25 amino acid, polybasicpeptide containing three disulfide bridges with a molecular weight of2639 daltons and a molecular formula of C₁₀₂H₁₇₂N₃₆O₃₂S₇.

Knottin proteins have a characteristic disulfide linked structure. Thisstructure is also illustrated in Gelly et al., “The KNOTTIN website anddatabase: a new information system dedicated to the knottin scaffold,”Nucleic Acids Research, 2004, Vol. 32, Database issue D156-D159. Atriple-stranded β-sheet is present in many knottins. The cysteinesinvolved in the knot are shown as connected by lines in FIG. 1Dindicating which Cys residues are linked to each other. The spacingbetween Cys residues is important in the present invention, as is themolecular topology and conformation of the engineered loop. Theengineered loop may contain RGD to provide an integrin binding loop.These attributes are critical for high affinity integrin binding. TheRGD mimic loop is inserted between knottin Cys residues, but the lengthof the loop must be adjusted for optimal integrin binding depending onthe three-dimensional spacing between those Cys residues. For example,if the flanking Cys residues are linked to each other, the optimal loopmay be shorter than if the flanking Cys residues are linked to Cysresidues separated in primary sequence. Otherwise, particular amino acidsubstitutions can be introduced that constrain a longer RGD-containingloop into an optimal conformation for high affinity integrin binding.

The present knottin proteins may contain certain modifications made totruncate the knottin, or to remove a loop or unnecessary cysteineresidue or disulfide bond.

The term “amino acid” includes both naturally-occurring and syntheticamino acids and includes both the D and L form of the acids as well asthe racemic form. More specifically, amino acids contain up to tencarbon atoms. They may contain an additional carboxyl group, andheteroatoms such as nitrogen and sulfur. Preferably the amino acids areα and β-amino acids. The term α-amino acid refers to amino acids inwhich the amino group is attached to the carbon directly attached to thecarboxyl group, which is the α-carbon. The term β-amino acid refers toamino acids in which the amino group is attached to a carbon one removedfrom the carboxyl group, which is the β-carbon. The amino acidsdescribed here are referred to in standard IUPAC single letternomenclature, with “X” meaning any amino acid.

The term “EETI” means Protein Data Bank Entry (PDB) 2ETI. Its entry inthe Knottin database is EETI-II. It has the sequence

(SEQ ID NO: 1) GC PRILMRCKQDSDCLAGCVCGPNGFCG.Full length EETI-II has a 30 amino acid sequence with a final proline atposition 30:

(SEQ ID NO: 2) 1 GC PRILMR  CKQDSDC LAGCVC GPNGF CGSP

Loops 1 and 3 are in bold and underlined. These loops can also be variedand affect binding efficiency, as is demonstrated below. Other loops maybe varied without affecting binding efficiency.

The term “AgRP” means PDB entry 1HYK. Its entry in the Knottin databaseis SwissProt AGRP_HUMAN, where the full-length sequence of 129 aminoacids may be found. It comprises the sequence beginning at amino acid87. An additional G is added to this construct. It also includes a C105Amutation described in Jackson, et al. 2002 Biochemistry, 41, 7565.

(SEQ ID NO: 3) GCVRLHESCLGQQVPCCDPCATCYC RFFNAF CYCR-KLGTAMNPCSRT

The dashed portion shows a fragment omitted in the “mini” version,below. The bold and underlined portion, from loop 4, is replaced by theRGD sequences described below. Loops 1 and 3 are shown between bracketsbelow:

(SEQ ID NO: 3) GC[VRLHES]CLGQQVPCC[DPCAT]CYCRFFNAFCYCR- KLGTAMNPCSRT

The term “mini” in reference to AgRP means PDB entry 1MRO. It is alsoSwissProt AGRP_HUMAN. It has the sequence, similar to that given above,

(SEQ ID NO: 4) GCVRLHESCLGQQVPCCDPAATCYC RFFNAF CYCRwhere the underlined “A” represents an amino acid substitution whicheliminates a possible dimer forming cystine. (Cystine herein refers tothe single amino acid; cysteine to the dimer.). The bold and underlinedportion, from loop 4, is replaced by the below described RGD sequences.

The term “agatoxin” means omega agatoxin PDB 1OMB and the SwissProtentry in the knottin database TOG4B_AGEAP. It has the sequence

(SEQ ID NO: 5) EDN--CIAEDYGKCTWGGTKCCRGRPCRC SMIGTN CECT- PRLIMEGLSFA

The dashes indicate portions of the peptide omitted for the “mini”agatoxin. An additional glycine is added to the N-terminus of themini-construct. The bold and underlined portion is replaced by the belowdescribed RGD sequences.

The term “loop domain” refers to an amino acid subsequence within apeptide chain that has no ordered secondary structure, and residesgenerally on the surface of the peptide. The term “loop” is understoodin the art as referring to secondary structures that are not ordered asin the form of an alpha helix, beta sheet, etc.

The term “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with at least 70% sequence identityto a reference sequence, preferably 80%, more preferably 85%, mostpreferably at least 90% or at least 95% sequence identity to thereference sequence over a specified comparison window, which in thiscase is either the entire peptide, a molecular scaffold portion, or abinding loop portion (˜9-11 residues). Preferably, optimal alignment isconducted using the homology alignment algorithm of Needleman and Wunsch(1970) J. Mol. Biol., 48:443 453. An indication that two peptidesequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Another indication for present purposes, that a sequence issubstantially identical to a specific sequence explicitly exemplified isthat the sequence in question will have an integrin binding affinity atleast as high as the reference sequence. Thus, a peptide issubstantially identical to a second peptide, for example, where the twopeptides differ only by a conservative substitution. “Conservativesubstitutions” are well known, and exemplified, e.g., by the PAM 250scoring matrix. Peptides that are “substantially similar” sharesequences as noted above except that residue positions that are notidentical may differ by conservative amino acid changes. As used herein,“sequence identity” or “identity” in the context of two nucleic acid orpolypeptide sequences makes reference to the residues in the twosequences that are the same when aligned for maximum correspondence overa specified comparison window. When percentage of sequence identity isused in reference to proteins it is recognized that residue positionswhich are not identical often differ by conservative amino acidsubstitutions, where amino acid residues are substituted for other aminoacid residues with similar chemical properties (e.g., charge orhydrophobicity) and therefore do not change the functional properties ofthe molecule. When sequences differ in conservative substitutions, thepercent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the NIH Multiplealignment workshop (http://helixweb.nih.gov/multi-align/).Three-dimensional tools may also be used for sequence comparison.

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “receptor tyrosine kinase” is used in its customary sense;examples are given below. The term “TAM receptor tyrosine kinase” refersto the TAM family of receptor kinases, including tyro3, Axl and MerTK.These are characterized by a conserved sequence within the kinase domainand adhesion molecule-like extracellular domains, and are describedfurther in Linger et al. “TAM receptor tyrosine kinases: biologicfunctions, signaling, and potential therapeutic targeting in humancancer,” Adv Cancer Res. 2008; 100:35-83.

GENERAL DESCRIPTION Engineering of Knottin Peptides

An important feature of the present fusion proteins is that the knottinportion is used for specific binding to a predetermined ligand. Theknottin binding is preferably engineered by replacing a native solventexposed loop with a short (e.g. 5-12 amino acid) sequence that has beenselected for binding to the predetermined ligand. The solvent-exposed(i.e. on the surface) loop will generally be anchored bydisulfide-linked cysteine residues. The new, or replacement amino acidsequence is preferably obtained by randomizing codons in the loopportion, expressing the engineered peptide, and selecting the mutantswith the highest binding to the predetermined ligand. This selectionstep may be repeated several times, taking the tightest binding proteinsfrom the previous step and re-randomizing the loops.

The EETI-II knottin peptide contains a disulfide knotted topology andpossesses multiple solvent-exposed loops that are amenable tomutagenesis. To evolve a binding interface with Gas6, we randomized thestructurally adjacent loops 1 and 3. Fusion of this EETI-II loop librarydirectly to the Axl Ig1 N-terminus (shown in FIG. 1D) did not perturbthe native Gas6-Axl interaction, which thereby resulted in a backgroundof tens of millions of single-digit nanomolar binders. The ability toisolate enhanced clones from such a background speaks to the power ofyeast surface display and quantitative fluorescent-activated cellsorting for protein engineering. Moreover, a starting library that doesnot suffer from loss-of-function differs with that of traditionaldirected evolution strategies, where random mutation to one of thebinding partners often results in decreased function for the majority ofthe initial library. Retention of wild-type properties in the domainaddition naïve library sheds light on natural evolutionary landscapes,whereby domain addition and evolution in nature may allow for theevolution of protein function without the cost of decreased activitywhile exploring sequence space.

A wide variety of knottin peptides may be used in the present fusionproteins. For example, when displayed on the yeast cell surface, thefollowing mutants bind to α_(v)β₃ integrin about 2-3× better than amutant with the RGD sequence from fibronectin.

TABLE 1 EETI sequences wherein the RGD motif (in italics in 1.4A)is found in the insert at positions 4-6. Peptide identifier SequenceSEQ ID NO: 1.4A GCAEPRGDMPWTWCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 6) 1.4BGCVGGRGDWSPKWCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 7) 1.4CGCAELRGDRSYPECKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 8) 1.4EGCRLPRGDVPRPHCKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 9) 1.4HGCYPLRGDNPYAACKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 10) 1.5BGCTIGRGDWAPSECKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 11) 1.5FGCHPPRGDNPPVTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 12) 2.3AGCPEPRGDNPPPSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 13) 2.3BGCLPPRGDNPPPSCKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 14) 2.3CGCHLGRGDWAPVGCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 15) 2.3DGCNVGRGDWAPSECKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 16) 2.3EGCFPGRGDWAPSSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 17) 2.3FGCPLPRGDNPPTECKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 18) 2.3GGCSEARGDNPRLSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 19) 2.3HGCLLGRGDWAPEACKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 20) 2.3IGCHVGRGDWAPLKCKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 21) 2.3JGCVRGRGDWAPPSCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 22) 2.4AGCLGGRGDWAPPACKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 23) 2.4CGCFVGRGDWAPLTCKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 24) 2.4DGCPVGRGDWSPASCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 25) 2.4EGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 26) 2.4FGCYQGRGDWSPSSCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 27) 2.4GGCAPGRGDWAPSECKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 28) 2.4JGCVQGRGDWSPPSCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 29) 2.5AGCHVGRGDWAPEECKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 30) 2.5CGCDGGRGDWAPPACKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 31) 2.5DGCPQGRGDWAPTSCKQDSDCRAGCVCGPNGFCG (SEQ ID NO: 32) 2.5FGCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (SEQ ID NO: 33) 2.5HGCPQGRGDWAPEWCKQDSDCPAGCVCGPNGFCG (SEQ ID NO: 34) 2.5JGCPRGRGDWSPPACKQDSDCQAGCVCGPNGFCG (SEQ ID NO: 35)

The above engineered knottins contain the RGD binding loop and bindspecifically to integrins, as described in copending application Ser.No. 12/418,376, filed Apr. 3, 2009. As described there, these loops maybe varied in the non-RGD residues to a certain degree without affectingbinding specificity and potency. For example, if three of the elevenresidues were varied, one would have about 70% identity to 2.5D. Theabove engineered knottins have been shown to bind specifically toα_(v)β₃, α_(v)β₅, and α₅β₁ integrins

Another example of a knottin peptide engineered to bind to integrins isAgRP. Table 2 below shows sequences of AgRP mutants isolated by flowcytometry and having an RGD sequence and flanking residues in loop 4, asindicated by the bolded residues:

TABLE 2 Sequences of additional AgRP mutants Clone Loop 4 sequence7A (5E) (SEQ ID NO: 36) GCVRLHESCLGQQVPCCDPAATCYCSGRGDNDLVCYCR7B (SEQ ID NO: 37) GCVRLHESCLGQQVPCCDPAATCYCKGRGDARLQCYCR7E (SEQ ID NO: 38) GCVRLHESCLGQQVPCCDPAATCYCVGRGDDNLKCYCR7J (6B) (SEQ ID NO: 39) GCVRLHESCLGQQVPCCDPAATCYCEGRGDRDMKCYCR7C (SEQ ID NO: 76) GCVRLHESCLGQQVPCCDPAATCYC YGRGDNDLR CYCR

Additional AgRP engineered knottins can be made as described in theabove-referenced US 2009/0257952 to Cochran et al. AgRP knottin fusionscan be prepared using AgRP loops 1, 2 and 3, as well as loop 4 asexemplified above.

Engineered Knottin Binding Partners

The engineered knottin is fused to another protein. The protein will tosome extent enter into the design of the engineered knottin according tothe present description. That is, the fusion partner and the knottinbinding partner will have a logical relationship in that they are in thesame biological pathway, they are directed to targets which may bebrought together to improve a therapeutic result, etc.

As exemplified below by an engineered knottin-tyrosine kinase receptorfusion, the fusion may be engineered to bind to a ligand for thetyrosine kinase. The fusion is administered and allowed to bind to theligand, thereby acting as a decoy to prevent the native ligand frombinding to the tyrosine kinase receptor. As further exemplified below,the entire tyrosine kinase receptor is not used; only portions that bindto a native ligand, preferably an agonist. In the case of Axl, the Ig1and Ig2 portions of the Axl receptor that bind to the Gas6 ligand areused. Gas 6, growth arrest-specific 6) belongs to the family of plasmavitamin K-dependent proteins. Gas 6 shares high structural homology withan anticoagulant protein, but has growth factor-like properties throughits interaction with receptor tyrosine kinases of the TAM family, tyro3,Axl and MerTK.

Another example of an engineered knottin-protein fusion is one where thefusion partner is a growth factor or active fragment of a growth factor,and the knottin is engineered to bind to endothelial cells such as maybe present in the vasculature or on tumors. This is exemplified by aknottin (AgRP) engineered to bind α_(v)β₃ integrins and a growth factoror growth factor fragment that binds to the Met receptor. Interactionbetween α_(v)β₃ integrin and extracellular matrix is crucial forendothelial cells sprouting from capillaries and for angiogenesis.Furthermore, integrin-mediated outside-in signals co-operate with growthfactor receptors to promote cell proliferation and motility. As anotherexample, Soldi et al., “Role of alphav beta3 integrin in the activationof vascular endothelial growth factor receptor-2,” The EMBO Journal(1999) 18, 882-892, reported that to determine a potential regulation ofangiogenic inducer receptors by the integrin system, they investigatedthe interaction between α_(v)β₃ integrin and tyrosine kinase vascularendothelial growth factor receptor-2 (VEGFR-2) in human endothelialcells. Both the VEGF receptor and the Met receptor (also known ashepatocyte growth factor receptor) are receptor tyrosine kinases.

Another example of binding partner selection is a fusion of anengineered knottin that binds to α_(v)β₃ integrin and NK1, a fragment ofthe polypeptide growth factor HGF/SF which acts as agonist of the METreceptor. As described below, NK1 was modified to create highly stable,more effective agonistic ligands, or modified to create highly stable,more effective antagonists.

EETI-Axl Fusions with a Synthetic Binding Domain (Through DomainAddition)

In the examples below, the Ecballium elaterium trypsin inhibitor II(EETI-II) serves as a synthetic binding domain to increase binding ofits fusion partner. EETI-II is a member of the knottin family ofpeptides which contain a characteristic interwoven disulfide-bondedframework that provides exquisite stability properties (FIG. 1B). Thesolvent exposed loops of EETI-II are tolerant to mutagenesis and havepreviously been individually engineered for novel recognitionproperties. However, in the present work, two structurally adjacentloops in EETI-II were concurrently randomized and the resulting libraryof EETI-II mutants was fused to wt Axl Ig1. Axl sequences are given inEntrez Gene Gene ID 558. This library was then screened to identifynovel EETI-Axl fusions with enhanced Gas6 binding affinity. That is,binding would occur through the Axl receptor and through the engineeredloops. We identified mutants with sub-nanomolar affinity following asingle round of directed evolution, wherein both engineered loops of theEETI-II mutant contributed to the enhanced affinity towards Gas6 throughthe creation of a novel binding face. This work supports domain additionand evolution for enhancing protein function, and also supports theEETI-II knottin as a scaffold for engineering novel recognitionproperties.

Domain Addition Library Design and Synthesis

To enhance the affinity of the Gas6/Axl interaction we fused a looplibrary of the EETI-II knottin peptide to the Axl Ig1 since the Ig1domain comprises the dominant binding site for Gas6. We chose a fusionto the Axl N-terminus because in the Gas6/Axl complex, the Axl Ig1N-terminus is in closer proximity to Gas6 than its C-terminus, and istherefore more likely to enable interaction of the EETI-II mutants withGas6 (FIG. 1C). Analysis of EETI-II and Axl structures shows fusion ofEETI-II to the Axl N-terminus would give approximately 11 amino acidspacing between tertiary structures of the two proteins. Therefore, wechose to directly fuse the EETI-II loop library to the Axl N-terminuswithout inclusion of additional linker residues. The final Pro30 residuein EETI-II and Pro20 of Axl Ig1 were excluded to improve the flexibilityof the linkage, resulting in EETI-II Ser29 fused directly to Axl Arg21.We chose EETI-II loops 1 and 3 for randomization as they arestructurally adjacent (FIG. 1B), which would allow for the formation ofa continuous binding face on the EETI-II knottin. Wild-type loops 1 and3 were concurrently replaced with randomized sequences of 7-10 and 6-8amino acids (FIG. 1D), respectively, using NNS codons. The NNS codonstrategy permits the inclusion of all 20 amino acids in the engineeredloops while limiting the frequency of stop codons by encoding for onlyone stop codon. Other degenerate library strategies could be employed.See, for other exemplary strategies, Kleeb et al., “Metabolicengineering of a genetic selection system with tunable stringency,”Proc. Nat. Acad. Sci. 104: 13907-13912 (2007).

Direct fusion was achieved by inclusion of an AvrII (C′CTAG,G) site,which encodes for a proline-arginine dipeptide, prior to Axl Ig1 aminoacid Gly22 in the yeast display pCT plasmid. The EETI-II loop librarywas designed to replace the first base pair of the restriction digestedAvrII site with a ‘T’, to give TCTAGG (SEQ ID NO: 40), which encodes forthe desired Ser-Arg linkage of EETI-II Ser29 and Axl Ig1 Arg21.

The cDNA for the EETI-II loop library was synthesized using standard PCRassembly techniques and the yeast display E-Axl library was generated byhomologous recombination to the pCT-Avr-Axl acceptor plasmid (SeeExamples). This library is hereto referred to as the E-Axl library; itcomprised 1.2×10⁸ individual transformants as determined by dilutionplating and colony counting. Sequence analysis of randomly selectedindividual clones confirmed intended fusion strategy, loop lengthdistribution, and a lack of mutation to the Axl Ig1 sequence.Approximately 30% of the clones contained full loop sequences withoutstop codons or mutations in line with previous reports of librariescontaining multiple randomized loops.

Identification of binding proteins from naïve libraries is challenging,in part due to the requirement that the affinity of the identifiedprotein must be high enough for detection. For example, in yeast surfacedisplay binding affinities in the single-digit μM range are below thelimits of detection and such proteins will generally not be enrichedduring library sorting. Domain addition and evolution can be used as an“anchoring” strategy, enabling identification of synthetic bindingdomains that enhance an existing interaction, but in isolation maythemselves possess affinity below the limits of detection. In support ofthis, the EETI-II mutants developed here exhibit weak binding affinitytowards Gas6 that are below the limits of detection when the knottinmutants are expressed in the absence of Axl. Subsequent affinitymaturation through traditional strategies or further domain addition andevolution can be used to generate fully synthetic binding agents withhigh affinity.

Library Screening and Sequence Analysis

Expression of the E-Axl library and its binding to Gas6 were assessed byimmunofluorescent labeling of the cmyc epitope tag on the yeast displayconstruct and the hexahistidine tag (SEQ ID NO: 77) on soluble Gas6,respectively (FIG. 2A). FIG. 2A shows the aga toxin component Aga 1p andAga 2p extending in that order from the yeast cell wall, as is known inyeast surface display. An anti-his antibody tagged with Hylite 448 22 isbound to the his tag 32 on Gas 6; the myc tag 26 is bound to a chickenanti-myc antibody 28, which in turn is bound by an anti-chicken antibodylabeled with Alexa 555, 30. A hemagglutinin tag is also included in thefusions. The Axl-Ig1 portion is fused to this, and binding of the Gas6ligand to the Axl is carried out. Strikingly, all members of thestarting library that expressed on the yeast cell surface bound to Gas6at the same levels as wild-type Axl Ig1 FIG. 2B). This demonstrates thatthe direct fusion of an EETI-II loop library to the Axl N-terminus doesnot perturb the native Gas6-Axl interaction. Consequently, this alsoresults in a background of tens of millions of wild-type, single-digitnanomolar binders from which rare improved clones must be separated.

For library screening using yeast surface display, often the top 1% ofbinding clones are collected; however, due to this extremely highbackground level of binding, we initially employed a conservative sortstrategy wherein the top 6% of binding clones was collected to decreaseprobability of losing rare clones with enhanced properties (FIG. 3).

FIG. 3 shows that when sorting the library, the first sorts wereconducted by screening for binding to soluble Gas6. Subsequent sortsused ‘off-rate’ sorts where binding to Gas6 was followed by incubationin the presence of excess competitor to impart selective pressure onenhanced kinetic dissociation. In the 6^(th) round of sorting weconducted a negative sort to clear mutants that were binding tosecondary anti-His antibody. Sort 6 products (below) show these werecompletely eliminated with a single round of sorting. Final sortproducts retained binding after a 46 h unbinding (‘off’) step.

To increase stringency in later sort rounds, ‘off-rate’ sorts wereconducted in which incubation with 2 nM Gas6 was followed by anunbinding step in the presence of a molar excess of soluble Axl receptorto serve as competitor. The excess competitor renders the dissociationof Gas6 from yeast-displayed E-Axl irreversible by sequestering freeGas6 in complex with soluble Axl receptor, thereby increasing theselective pressure for clones with slower dissociation rate.

Bispecific Proteins that Target Integrin and a Growth Factor Receptor

Described in Example 8 is the preparation of a fusion between a knottin(AgRP) engineered to bind α_(v)β₃ integrins, and a fragment comprisingthe N-terminal and first kringle domains of HGF (termed NK1). Thisportion of HGF (hepatocyte growth factor) binds to the Met receptor.c-Met (MET or MNNG HOS Transforming gene) is a proto-oncogene thatencodes a protein known as hepatocyte growth factor receptor (HGFR). Thehepatocyte growth factor receptor protein possesses tyrosine-kinaseactivity. The primary single chain precursor protein ispost-translationally cleaved to produce the alpha and beta subunits,which are disulfide linked to form the mature receptor.

The α_(v)β₃ integrin receptor is over-expressed on many solid tumorcells making it an important cancer target. The Agouti related protein(AgRP), a cystine-knot peptide, contains four disulfide bonds and foursolvent-exposed loops. It was engineered to target α_(v)β₃ integrinreceptors with pM binding affinity. The AgRP mutant, 7A, was shown tohave the tightest binding affinity. The K_(D) values of the 7A mutantagainst U87MG and K562-α_(v)β₃ cells are 0.78 nM and 0.89 nM,respectively.

The Met receptor tyrosine kinase and its ligand hepatocyte growth factor(HGF) play an important role in mediating both tumor progression andtissue regeneration. The N-terminal and first kringle domains (NK1) ofHGF is a naturally occurring splice variant that retains the ability toactivate the Met receptor. However, NK1 is a weak agonist and isrelatively unstable, limiting its therapeutic potential. We engineeredNK1 mutants that function as Met receptor agonists and antagonists andpossess enhanced biochemical and biophysical properties. As describedbelow, we first evolved NK1 for enhanced stability and recombinantexpression yield using yeast surface display. The NK1 mutants isolatedfrom our library screens functioned as weak Met receptor antagonists,due to mutation of a residue which mediates NK1 homodimerization. Weintroduced point mutations that restored this NK1 homodimerizationinterface to create an agonistic ligand, or that further abolished theseinteractions to create more effective antagonists. The best antagonistsexhibited melting temperatures up to ˜64° C., a 15° C. improvement overantagonists derived from wild-type NK1, and up to a 40-fold improvementin expression yield.

The crosstalk between integrin and c-Met signaling pathways was studiedand showed a significant relationship. The signal transduction ofHGF/SF, the natural ligand of Met receptors, can induce ligand-bindingactivity in functionally-inactive α_(v)β₃ integrins in epithelial andendothelial cells. Therefore, a dual-specific protein that targets andinhibits both α_(v)β₃ integrin and Met receptors has promise as aneffective cancer therapeutic, especially compared to single receptortargeting agents.

Receptor Tyrosine Kinase (“RTK”) Fragments Useful in Fusions

The present fusion proteins may include a variety of receptor tyrosinekinases. These proteins have been well characterized as to theirextracellular and ligand-binding motifs. They include RTK class I (EGFreceptor family) (ErbB family); RTK class II (Insulin receptor family);RTK class III (PDGF receptor family); RTK class IV (FGF receptorfamily); RTK class V (VEGF receptors family); RTK class VI (HGF receptorfamily); RTK class VII (Trk receptor family); RTK class VIII (Ephreceptor family); RTK class IX (AXL receptor family); RTK class X (LTKreceptor family); RTK class XI (TIE receptor family); RTK class XII (RORreceptor family); RTK class XIII (DDR receptor family); RTK class XIV(RET receptor family); RTK class XV (KLG receptor family); RTK class XVI(RYK receptor family); and RTK class XVII (MuSK receptor family).Preferably, in preparing fusion proteins with these receptors, one wouldprepare a polypeptide containing only a portion of the receptor, i.e.containing the extracellular N-terminal region, which exhibits a varietyof conserved elements including immunoglobulin (Ig)-like or epidermalgrowth factor (EGF)-like domains, fibronectin type III repeats, orcysteine-rich regions that are characteristic for each subfamily ofRTKs; these domains contain primarily a ligand-binding site, which bindsextracellular ligands, e.g., a particular growth factor or hormone. Theintracellular C-terminal region displays the highest level ofconservation and comprises catalytic domains responsible for the kinaseactivity of these receptors, which catalyses receptorautophosphorylation and tyrosine phosphorylation of RTK substrates.

Receptor tyrosine kinase sequences are available from a variety ofsources, including Genbank. Exemplary sequences that may be used tocreate fragments and fusion proteins according to the present inventionare given, e.g. in Rand et al., “Sequence survey of receptor tyrosinekinases reveals mutations in glioblastomas.” Proc. Nat. Acad. Sci. Oct.4, 2005 vol. 102 no. 40 14344-14349. The following list is taken fromthat publication.

Genbank Accession Number RTK Description NM_004439 Ephrin type-Areceptor 5 precursor NM_001982 Receptor tyrosine-protein kinase erbB-3precursor NM_020975 Proto-oncogene tyrosine-protein kinase receptor retprecursor NM_002944 Proto-oncogene tyrosine-protein kinase ROS precursorNM_002530 NT-3 growth factor receptor precursor NM_002019 Vascularendothelial growth factor receptor 1 precursor NM_005012Tyrosine-protein kinase transmembrane receptor ROR1 precursor NM_004560Tyrosine-protein kinase transmembrane receptor ROR2 precursor NM_004304ALK tyrosine kinase receptor precursor NM_000222 Mast/stem cell growthfactor receptor precursor NM_006180 BDNF/NT-3 growth factors receptorprecursor NM_006206 Alpha platelet-derived growth factor receptorprecursor NM_004441 Ephrin type-B receptor 1 precursor NM_000875Insulin-like growth factor I receptor precursor NM_004438 Ephrin type-Areceptor 4 precursor NM_000208 Insulin receptor precursor NM_004119 FLcytokine receptor precursor NM_006182 Discoidin domain receptor 2precursor NM_000141 Fibroblast growth factor receptor 2 precursorNM_023110 Basic fibroblast growth factor receptor 1 precursor.

See also, Lee et al., “Vascular endothelial growth factor-relatedprotein: a ligand and specific activator of the tyrosine kinase receptorFlt4,” PNAS Mar. 5, 1996 vol. 93 no. 5 1988-1992.

The exact fragment of the receptor to be used in the present inventioncan be determined in view of the present teachings and existingknowledge of receptor structure. It is not necessary that an exactsequence that encodes only the ligand binding pocket be used. Someflexibility to include additional amino acids is tolerated. For example,as disclosed in US 20040132634, The N-terminal extracellular region ofall Eph family members contains a domain necessary for ligand bindingand specificity, followed by a cysteine-rich domain and two fibronectintype II repeats. In general, the N terminal portion, of about 400, 500or 600 amino acids may be used as a ligand binding fragment of areceptor tyrosine kinase.

The above listings provide amino acid and nucleotide sequences. Othernucleotide sequences may be obtained from Genbank by searching on thename of the peptide or protein. Knottin DNA sequences may be obtainedfrom the given amino acid sequences, using any codon assignment; codonassignment may be selected based on the expression vector used, such asyeast. An EETI nucleotide sequence is given in WO0234906, GenBankAX497055; an AGRP nucleotide sequence may be found at NG_(—)011501; anagatoxin nucleotide sequence may be found at Genbank M95540.1. Anotherknottin amino acid and nucleic acid sequence may be found in J.Microbiol. Biotechnol. (2010), 20(4), 708-711, relating to the knottinPsacotheasin.

Receptor Ligand Fragments Useful in Fusions

Exemplified here are the particular receptor ligands hepatocyte growthfactor and the antibody Fc fragment. The hepatocyte growth factor (alsotermed c-met) was fragmented to yield the portion of it that is known tobind to the met receptor. This fragment of HGF is known as the NK1fragment. An exemplary sequence is given in SEQ ID NO: 66. This sequencecontains portions of sequences in the PAN_Apple super family and of theKR superfamily. Therefore, one would expect that the presentlyexemplified compositions, given the present teachings, could be expandedto include hepatocyte growth factor-like proteins; pplasminogen domaincontaining proteins; macrophage stimulating factor 1; and otherplasminogen-related growth factors such as RON (“recepteur d′ origineNantais”). See, Maestrini et al., “A family of transmembrane proteinswith homology to the MET-hepatocyte growth factor receptor,” PNAS Jan.23, 1996 vol. 93 no. 2 674-678. Also, in mammals, hepatocyte growthfactor is a homolog of serine proteases but it has lost its proteolyticactivity.

Administration of Bispecific Proteins

The present fusion proteins may be administered in vitro, such as incell culture studies, or to cells intended for transplant, but may alsobe administered in vivo. A variety of formulations and dosing regimentsused for therapeutic proteins may be employed. The pharmaceuticalcompositions may contain, in addition to the CFP, suitablepharmaceutically acceptable carriers, biologically compatible vehiclesand additives which are suitable for administration to an animal (forexample, physiological saline) and eventually comprising auxiliaries(like excipients, stabilizers or diluents) which facilitate theprocessing of the active fusion proteins into preparations which can beused pharmaceutically. Such compositions can be eventually combined withanother therapeutic composition acting synergically or in a coordinatedmanner with the chimeric proteins of the invention. Alternatively, theother composition can be based with a fusion protein known to betherapeutically active against the specific disease (e.g. herceptin forbreast cancer). Alternatively, the pharmaceutical compositionscomprising the soluble can be combined into a “cocktail” for use in thevarious treatment regimens.

The pharmaceutical compositions may be formulated in any acceptable wayto meet the needs of the mode of administration. For example, the use ofbiomaterials and other polymers for drug delivery, as well the differenttechniques and models to validate a specific mode of administration, aredisclosed in literature (Luo B and Prestwich G D, 2001; Cleland J L etal., 2001).

Any accepted mode of administration can be used and determined by thoseskilled in the art to establish the desired blood levels of then fusionprotein. For example, administration may be by various parenteral routessuch as subcutaneous, intravenous, epidural, topical, intradermal,intrathecal, direct intraventricular, intraperitoneal, transdermal (e.g.in slow release formulations), intramuscular, intraperitoneal,intranasal, intrapulmonary (inhaled), intraocular, oral, or buccalroutes.

Other particularly preferred routes of administration are aerosol anddepot formulation. Sustained release formulations, particularly depot,of the invented medicaments are expressly contemplated.

Parenteral administration can be by bolus injection or by gradualperfusion over time. Preparations for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions,which may contain auxiliary agents or excipients known in the art, andcan be prepared according to routine methods. In addition, suspension ofthe active fusion proteins as appropriate oily injection suspensions maybe administered. Suitable lipophilic solvents or vehicles include fattyoils, for example, sesame oil, or synthetic fatty acid esters, forexample, sesame oil, or synthetic fatty acid esters, for example, ethyloleate or triglycerides. Aqueous injection suspensions that may containsubstances increasing the viscosity of the suspension include, forexample, sodium carboxymethyl cellulose, sorbitol, and/or dextran.Optionally, the suspension may also contain stabilizers. Pharmaceuticalcompositions include suitable solutions for administration by injection,and contain from about 0.01 to 99 percent, preferably from about 20 to75 percent of active fusion protein together with the excipient.Compositions that can be administered rectally include suppositories.

For parenteral (e.g. intravenous, subcutaneous, intramuscular)administration, the active protein(s) can be formulated as a solution,suspension, emulsion or lyophilised powder in association with apharmaceutically acceptable parenteral vehicle (e.g. water, saline,dextrose solution) and additives that maintain is otonicity (e.g.mannitol) or chemical stability (e.g. preservatives and buffers). Theformulation is sterilized by commonly used techniques. For transmucosaladministration, penetrants appropriate to the barrier to be permeatedare used in the formulation. Such penetrants are generally known in theart.

Pharmaceutical or physiologically acceptable preparations that can betaken orally include push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with fillers such as lactose, binders such as starches, and/orlubricants such as talc or magnesium stearate and, optionally,stabilizers. In soft capsules, the active fusion proteins may bedissolved or suspended in suitable liquids, such as fatty oils, liquidparaffin, or liquid polyethylene glycols. In addition, stabilizers maybe added. All formulations for oral administration should be in dosagessuitable for such administration.

The fusion proteins may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder or lyophilized form for constitution with asuitable vehicle, such as sterile pyrogen-free water, before use.

In addition to the formulations described previously, the fusionproteins may also be formulated as a depot preparation. Such long actingformulations may be administered by implantation (for examplesubcutaneously or intramuscularly) or by intramuscular injection. Thus,for example, the fusion proteins may be formulated with suitablepolymeric or hydrophobic materials (for example as an emulsion in anacceptable oil) or ion exchange resins, or as sparingly solublederivatives, for example, as a sparingly soluble salt. Additionally, thefusion proteins may be delivered using a sustained release system, suchas semipermeable matrices of solid hydrophobic polymers containing thetherapeutic agent. Various sustained-release materials have beenestablished and are well known by those skilled in the art. Sustainedrelease capsules may, depending on their chemical nature, release thefusion proteins for a few weeks up to over 100 days or one year.

It is understood that the dosage administered will be dependent upon theage, sex, health, and weight of the recipient, kind of concurrenttreatment, if any, frequency of treatment, and the nature of the effectdesired. The dosage will be tailored to the individual subject, as isunderstood and determinable by one of skill in the art. The total doserequired for each treatment may be administered by multiple doses or ina single dose. The pharmaceutical composition of the present inventionmay be administered alone or in conjunction with other therapeuticsdirected to the condition, or directed to other symptoms of thecondition. Usually a daily dosage of active protein is comprised between0.01 to 100 milligrams per kilogram of body weight. Ordinarily 1 to 40milligrams per kilogram per day given in divided doses or in sustainedrelease form is effective to obtain the desired results. Second orsubsequent administrations can be performed at a dosage, which is thesame, less than, or greater than the initial or previous doseadministered to the individual. According to the invention, thesubstances of the invention can be administered prophylactically ortherapeutically to an individual prior to, simultaneously orsequentially with other therapeutic regimens or agents (e.g. multipledrug regimens), in a therapeutically effective amount. Active agentsthat are administered simultaneously with other therapeutic agents canbe administered in the same or different compositions.

For any protein used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Forexample, a dose can be formulated in animal models to achieve acirculating concentration range that includes or encompasses aconcentration point or range shown to decrease cytokine expression in anin vitro system. Such information can be used to more accuratelydetermine useful doses in humans. A therapeutically effective doserefers to that amount of the fusion protein that results in ameliorationof symptoms in a patient. Toxicity and therapeutic efficacy of suchfusion proteins can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals, e.g., for determining theLD50, (the dose lethal to 50% of the test population) and the ED50 (thedose therapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio between LD50 and ED50. Fusion proteinsthat exhibit high therapeutic indices are preferred. The data obtainedfrom these cell culture assays and animal studies can be used informulating a range of dosage for use in humans. The dosage of suchfusion proteins lies preferably within a range of circulatingconcentrations that include the ED50, with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. The exactformulation, route of administration and dosage can be chosen by theindividual physician in view of the patient's condition.

EXAMPLES

As described in Examples 1 through 5, we have developed a generalapproach to engineering existing protein-protein interactions we referto as “domain addition and evolution” in which enhancement isaccomplished by expanding the binding interface through the addition andsubsequent in vitro evolution of a synthetic binding domain. We validatethis approach by showing the ability to enhance the native high affinityligand-receptor interaction between Gas6 and the Axl receptor throughaddition and evolution of a synthetic knottin binding domain.

We identified EETI-II-axl fusion mutants with up to 4-fold enhancedaffinity towards Gas6. Importantly, Axl Ig1 did not accumulate mutationsduring the mutagenesis and screening process, indicating that theenhancement in affinity can be attributed to the engineered EETI-IImutants. Individual reversion of the engineered loops to wild-typeEETI-II sequence confirmed some EA mutants require both engineered loopsfor the enhanced affinity. To our knowledge, this is the first instanceof engineering two loops of a knottin into a binding face towards anexogenous target. Also, the three EA mutants each comprise approximately45% non-native EETI-II amino acid sequence. Together, this furthervalidates the robust nature of the knottin fold for generating novelbinding reagents. This work is also relevant given the role of Axl incancer metastasis. Dominant negative Axl receptors suppress tumor cellmigration and metastasis (Vajkoczy et al., 2006; Rankin et al., 2010),and the enhanced affinity EA mutants may be useful therapeuticcandidates.

An additional application of this approach includes identification ofbinding proteins from naïve libraries. EETI-II peptides engineered forbinding tumor targets hold significant promise for in vivo molecularimaging applications. However, identification of binding proteins fromnaïve libraries is challenging, in part due to the requirement that theaffinity of the identified protein must be high enough for detection.For example, in yeast surface display binding affinities in thesingle-digit μM range are below the limits of detection and suchproteins will generally not be enriched during library sorting. Domainaddition and evolution can be used as an “anchoring” strategy, enablingidentification of synthetic binding domains that enhance an existinginteraction, but in isolation may themselves possess affinity below thelimits of detection. In support of this, the EETI-II mutants developedhere exhibit weak binding affinity towards Gas6 that are below thelimits of detection when the knottin mutants are expressed in theabsence of Axl. Subsequent affinity maturation through traditionalstrategies or further domain addition and evolution can be used togenerate fully synthetic binding agents with high affinity.

As described below, the engineered EETI knottin variant 2.5D, whichbinds to αvβ3/αvβ5 integrin was directly fused to the N-terminus of wtAxl Ig1. The concept of this multi-specific fusion protein was validatedusing yeast-surface display by showing that EETI 2.5D-Axl bound to αvβ3integrin and Gas6 at levels comparable to the mono-specific proteinsEETI 2.5D and Axl, respectively. Furthermore, binding of αvβ3 integrinor Gas6 was not affected by the presence of a saturating concentrationof the other target, suggesting EETI 2.5D-Axl is capable ofsimultaneously interacting with both αvβ3 integrin and Gas6. The EETI2.5D-Axl fusion protein was able to be produced recombinantly inmicrobial hosts on a scale of 35 mg/L. The resulting protein displayedhigh affinity (K_(D)˜2 nM) to αvβ3 integrin expressed on the cellsurface.

Example 1 Reagents and Media

SD-CAA media contained 20 g/L glucose, 6.7 g/L yeast nitrogen basewithout amino acids, 5.4 g/L Na2HPO4, 8.6 g/L NaH2PO4.H2O, and 5 g/LBacto casamino acids; SG-CAA media was identical, except glucose wassubstituted with 20 g/L galactose. SD-CAA pH 4.5 media was identical toSD-CAA, except phosphates were replaced with 13.7 g/L sodium citratedihydrate, 8.4 g/L citric acid anhydrous, and adjusted to pH 4.5. Gas6and Axl-Fc proteins were purchased from R&D Systems, chicken anti-cmycand goat anti-chicken Alexa 555 antibodies were purchased fromInvitrogen, mouse anti-His Hilyte Fluor 488 monoclonal antibody waspurchased from Anaspec. Phosphate buffered saline (PBS) is composed of11.9 mM sodium phosphate pH 7.4, 137 mM sodium chloride, 2.7 mMpotassium chloride. PBS/BSA consisted of PBS with 1 mg/mL bovine serumalbumin.

Example 2 Yeast Surface Display Library Generation: EETI/Axl Fusions

Four forward assembly primers replacing EETI-II loop 1 with 7, 8, 9 or10 degenerate NNS codons and three reverse assembly primers replacingEETI-II loop 3 with 6, 7 or 8 degenerate NNS codons was used to assemblethe EETI-II loop library. (EETI-II amino acid sequence is GenbankAccession No. P12071; DBNA sequences are given in copending U.S. Ser.No. 12/418,376, filed Apr. 3, 2009, also incorporated by reference. DNAsequences may designed as desired by reverse translation of the aminoacid sequences given.) The primer sequences were complementary to theregions adjacent the loops. The amino acid sequences of EETI-II and therandomized loop 1 and loop 3, as well as the loops randomized in Axl areshown in FIG. 1D. The four forward primers were pooled and used at 1 μMeach, and each of the three reverse primers were pooled and used at 1.33μM each, with 1×KOD polymerase buffer, 0.2 mM dNTPs, 1.5 mM MgCl₂, 1 MBetaine, and 2.5 units KOD polymerase (Novagen). Thermocyling parameterswere: Step 1-95° C. for 2 min; Step 2-95° C. for 30 sec, 55° C. for 30sec, 72° C. for 1 min (30 cycles); Step 3-72° C. for 10 min. AssembledDNA (0.6 μL) was amplified using 2 μM forward and reverse amplificationprimers, 1×Pfx50 buffer, 0.2 mM dNTPs, and 5 units Pfx50 DNA polymerase(Invitrogen). Forward amplification primer had 50 bp homology to the pCTbackbone, while the reverse amplification primer contained 50 bphomology to the Axl Ig1 N-terminus and was designed to ensureappropriate Ser-Arg linkage of the EETI-II C-terminus (Ser29) with theAxl Ig1 N-terminus (Arg21). For preparation of the plasmid backbone, Axlamino acids comprising the Ig1 domain (22-132; Genbank Accession NO.P30530) were cloned into the yeast display pCT plasmid (Boder andWittrup, 1997) using NheI and BamHI restriction sites. We included anAvrII restriction site directly 5′ of the Axl sequence. This was placeddownstream of the NheI site with a 14 bp spacer of “junk DNA” tofacilitate restriction digest. We termed this plasmid pCT Avr-Axl.Plasmid backbone for library synthesis was generated by digesting pCTAvr-Axl with NheI and AvrII restriction enzymes. A total of ˜50 μg ofcDNA insert and ˜25 μg of restriction digested pCT Avr-Axl backbone wastransformed into EBY100 by electroporation and assembled in vivo byhomologous recombination. A library of 1.2×10⁸ transformants wasobtained, as estimated by serial dilution plating and colony counting.Sequence analysis of randomly selected clones confirmed appropriatefusion of EETI-II with Axl Ig1 and desired loop length distribution inthe EETI-II mutants.

Yeast surface display is described further in U.S. Pat. No. 6,423,538.Generally, at least 10⁴ transformants will be obtained.

Primers were designed as follows:

DNA Oligonucleotide Primers for EETI-Axl Library Synthesis/Assembly andAmplification

In the sequences below, the nucleotides used for homology to the plasmidbackbone are shown at the 5′ end up to the first slash. The part of theprimer between the first slash and the double slash and the triple slashand the 3′ end correspond to residues of EETI-II. N stands for anynucleotide and S is a mixture of G and C. The part of the primer betweenthe double slash and the triple slash are nucleotides used to producerandomized residues for EETI-II loop 1 or loop 3.

L1_7X_fwd: (SEQ ID NO: 67) Ggttctgctagc/ggttgt//nnsnnsnnsnnsnnsnnsnns///tgtaaacaagattctgattgtttggctggttgtgtt L1_8X_fwd: (SEQ ID NO: 68)Ggttctgctagc/ggttgt//nnsnnsnnsnnsnnsnnsnnsnns///tgtaaacaagattctgattgtttggctggttgtgtt L1_9X_fwd: (SEQ ID NO: 69)Ggttctgctagc/ggttgt//nnsnnsnnsnnsnnsnnsnnsnnsnns///tgtaaacaagattctgattgtttggctggttgtgtt L1_10X_fwd: (SEQ ID NO: 70)Ggttctgctagc/ggttgt// nnsnnsnnsnnsnnsnnsnnsnnsnnsnns///tgtaaacaagattctgattgtttggctggttgtgtt

In the case of the reverse primers below, the 5′ end up to the firstslash was homologous to nucleotides encoding the N terminus of the Axlreceptor construct, which is also part of the acceptor plasmid backbone.As above, the region between the first slash and the double slash andthe triple slash and the 3′ end correspond to residues of EETI-II. Nstands for any nucleotide and S is a mixture of G and C.

L3_6X_rev: (SEQ ID NO: 71) Cgtgcccct/gagaccaca//snnsnnsnnsnnsnnsnn///acaaacacaaccagccaaacaatcag L3_7X_rev: (SEQ ID NO: 72)Cgtgcccct/gagaccaca//snnsnnsnnsnnsnnsnnsnn/// acaaacacaaccagccaaacaatcagL3_8X_rev: (SEQ ID NO: 73)Cgtgcccct/gagaccaca//snnsnnsnnsnnsnnsnnsnnsnn///acaaacacaaccagccaaacaatcag

After library synthesis by PCR assembly, the library was amplified usingthe amplification primers below, which contain ˜50 base pairs ofhomology to the plasmid backbone (underlined, which comprises homologyto the Axl sequence for the case of the reverse amplification primer).The ˜50 base pairs of homology allows for assembly of the library insertand plasmid backbone as described by “Raymond C K, Pownder T A, Sexson SL. 1999. General method for plasmid construction using homologousrecombination. Biotechniques 26:134-138, 140-131.”

Library_amplification_reverse: (SEQ ID NO: 74)Ttccctgggttgcccacgaagggactttcttcagcctgcgtgcccct/ gctaccacaLibrary_amplification_forward: (homologyto plasmid backbone portion is 5′ of slash) (SEQ ID NO: 75)Ggtggttctggtggtggtggttctggtggtggtggttctgctagc/ ggttgt

Example 3 Library Screening with Gas6

Various concentrations of Gas6 were incubated with yeast-displayedlibraries in PBS/BSA for ˜2-3 hr at room temperature. For the finalhour, chicken anti-cmyc antibodies were added to a final dilution of1:250. Cells were pelleted by centrifugation, washed with 1 mL ice coldPBS/BSA, and resuspended in PBS/BSA containing 1:100 dilution of goatanti-chicken A555 and 1:100 dilution of mouse anti-His 488 antibodiesfor 25 min on ice. Cells were pelleted, washed with 1 mL ice coldPBS/BSA, and sorted by fluorescence-activated cell sorting (FACS) on aVantage SE flow cytometer (Stanford FACS Core Facility). Collected cellswere amplified in SD-CAA pH 4.5 media and induced for expression inSG-CAA media at 30° C. for additional rounds of FACS to yield anenriched pool of mutants. The first round of sorting by FACS consistedof three separate sorts for a total of approximately 8×10⁷ sorted cells,while subsequent sort rounds analyzed at least 10× the number of yeastcollected in the previous round to ensure sufficient sampling ofremaining library diversity. Sort stringency was increased by decreasingthe concentration of Gas6. In the later sort rounds, followingincubation with Gas6 cells were pelleted, washed, and incubated in thepresence of excess competitor (˜50-fold molar excess of Axl-Fc) for“off-rate” sorts. In the final hour of the unbinding step chickenanti-cmyc was added to 1:250 final dilution. Cells were pelleted,washed, and stained with secondary antibodies as above. Plasmid DNA wasrecovered from yeast cultures using a Zymoprep kit (Zymo Research) andtransformed into XL-1 blue supercompetent E. coli cells (Stratagene) forplasmid miniprep. DNA sequencing was performed by MC Lab (South SanFrancisco, Calif.).

After five rounds of sorting, the library began to enrich for clonespossessing stronger binding than wild-type Axl Ig1 (FIG. 3). A commonproblem in screening libraries containing randomized sequences is thepotential to screen for artifactual binders. For example, since we areilluminating Gas6 binding using an anti-hexahistidine secondary antibody(“hexahistidine” disclosed as SEQ ID NO: 77), some of the “enhanced”clones actually bound to the secondary antibody. To control for this, weconducted a negative sort with 0 nM Gas6 and secondary antibody labelingas usual to clear secondary binders from the collected pool (FIG. 3,Sort 6). We continued to monitor for secondary binders, but this singlenegative sort was sufficient for eliminating artifactual binders fromall subsequent sort products. Ultimately, we obtained an enriched poolof mutants with enhanced binding to Gas6 over wild-type Axl Ig1. Forcomparison, the final sort, which used a 46 h ‘off’ step, exhibitedhigher persistent binding than the fourth sort, which only used a 4 h‘off’ step, demonstrating significant improvement in kineticdissociation rate.

Example 4 Characterization of Engineered Mutants

Gas6 (0.05-400 nM) was added to 5×10⁴ yeast cells displaying protein ofinterest in PBS/BSA at room temperature, using volumes, cell numbers,and incubation times experimentally determined to avoid ligand depletionand reach binding equilibrium. Cells were pelleted and washed with icecold PBS/BSA and resuspended in PBS/BSA containing 1:250 dilution ofchicken anti-cmyc and incubated on ice for 40 min. Cells were pelleted,washed and resuspended in PBS/BSA containing a 1:100 dilution of goatanti-chicken and mouse anti-His secondary antibodies for 20 min on ice.Cells were washed and analyzed using a FACSCalibur flow cytometer(Becton Dickinson) and FlowJo software (Treestar, Inc). Bindingtitrations were fit to a four-parameter sigmoidal curve usingKaleidagraph software to determine the equilibrium binding constant(K_(D)). For kinetic unbinding tests cells were incubated with 2 nM Gas6until binding equilibrium was reached, then were washed, pelleted, andincubated in the presence of 50-fold molar excess Axl-Fc as describedabove for off-rate sorts for 0, 1, 4, 9.25, 23, or 46 hrs. Persistentbinding was analyzed by flow cytometry and unbinding was fit to a singleor double exponential decay curves as appropriate using Kaleidographsoftware. Persistent binding for reversion to wild-type EA loop variantswas conducted identically to the kinetic binding tests, except unbindingstep was conducted for 0-9.25 hrs.

Sequencing a total of 31 randomly selected clones from products of the7^(th), 8^(th) and 9^(th) rounds of sorting revealed twelve uniqueclones, with a 10^(th) round of sorting enriching for two of the clonesfrom the 9^(th) round sort products (Table 3, below). All clonesexhibited loop lengths in line with the initial library design and noclones contained mutations in the Axl sequence, indicating the enhancedaffinity of EA clones is specific to the EETI-II mutants. Three of thetwelve clones contained a PGM motif in loop 3, with two additionalclones containing either PTM or PGK, for a common P-G/T-M/K motif. Therewas also lesser occurrence an L or L-X preceding and R-S succeeding theP-G/T-M/K motif (FIG. 1D). Interestingly, only four of the twelve EAmutants, EA 7.01, EA 7.05, EA7.06, and EA 8.04, did not containcysteines in the engineered loops, but one of these, EA 7.05, containeda cys to arg mutation in the conserved cysteine residue precedingloop 1. Some mutants containing the P-T/G-M/K motif in loop 3 alsocontained a cysteine in an engineered loop, suggesting the additionalcysteines may not completely perturb the EETI-II loop structure (Table3). However, to minimize potential effects of unpaired cysteines, EA7.01, 7.06, and 8.04 were selected for further investigation. Forbrevity, the entire sequences of the Axl fusions is not given here,although are set forth in the attached sequence listing for SEQ ID NOs:41, 46 and 50. It is understood that the Axl Ig1 sequence is set forthbelow in both native and mutated forms and is used in the EA sequencesbelow in native form, except where noted. For example, EA 7.01 as listedin Table 3 is fused to the N terminal of Axl Ig1 continues with the Nterminal sequence of the Axl Ig1 sequence, as shown in FIG. 1D and inSEQ ID NO: 41. The other EAs listed in table 3 are similarly fused withthe Axl sequence beginning “RGT . . . ”. Full length sequences are givenin SEQ ID NOs: 41, 46 and 50, illustrated in FIG. 1D up to the ‘QAE . .. ” portion. To reiterate, in the polypeptides of Table 3 below, theterminal GS is fused to the Axl Ig1 domain as shown in SEQ ID NO: 84,below.

TABLE 3 Sequences of EA products from final sort rounds #AA #AA SEQ IDClone* AA sequence L1** L3** #rpt NO: Notes Wt GC PRILMR CKQDSDCLAGCVC 65 2 EETI-II GPNGF CGSP EA 7.01 GC ALMTPSAVD 9 6 2 ResiduesCKQDSDCLAGCVC LPGMVR 1-33 of CGS SEQ ID NO: 41 EA 7.02 GC LGNVRACVSV6, 10 7, 8 1 42 CKQDSDCLAGCVC ELARSNK CCGS EA 7.03 GC TAVRPCT 5, 7 8 143 CKQDSDCLAGCVC TLLPGMLM CGS EA 7.04 GC WPRVSCVLWH 5, 10 8 1 44CKQDSDCLAGCVC ILTRHKTV CGS EA 7.05 GR RWWTLAR ‘7’ 8 1 45CKQDSDCLAGCVC ILDPGKRS CGS EA 7.06 GC LGGVALAH 8 6 1 ResiduesCKQDSDCLAGCVC HILPEL CGS 1-32 of SEQ ID NO: 46 EA 7.08 GC HENGLPLI 85, 7 1 47 CKQDSDCLAGCVC SSHNWCQ CGS EA 8.01 GC ALMTPSAVD 9 6 6 48Same as CKQDSDCLAGCVC LPGMVR 7.01 CGS EA 8.02 GC VCLCCGPSGS ??, 10 7 349 CKQDSDCLAGCVC AANHKDN CGS EA 8.04 GC SWSTLAR 7 8 2 ResiduesCKQDSDCLAGCVC MLEPGMRS 1-33 of CGS SEQ ID NO: 50 EA 8.05 GC WLECWYR 3, 75, 8 3 51 CKQDSDCLAGCVC YLCPTMGS CGS EA 8.08 GC LGNVRACVSV 6, 10 7, 8 152 Same as CKQDSDCLAGCVC ELARSNK 7.02 CCGS EA GC VRVASHLWF 9 5, 6 3 539.01⁺ CKQDSDCLAGCVC CGRPNV CGS EA 9.02 GC VCLCCGPSGS ??, 10 7 2 54Same as CKQDSDCLAGCVC AANHKDN 8.02 CGS EA 9.05 GC CSLRWCVSRV ??, 10 7 255 CKQDSDCLAGCVC INPNKPL CGS EA 9.07 GC ALMTPSAVD 9 6 1 56 Same asCKQDSDCLAGCVC LPGMVR 7.01 CGS EA GC VRVASHLWF 9 5, 6 2 57 Same as 10.01*CKQDSDCLAGCVC CGRPNV 9.01 CGS EA GC CSLRWCVSRV ??, 10 7 6 58 Same as10.02 CKQDSDCLAGCVC INPNKPL 9.05 CGS *Randomly selected clones fromproducts of 7^(th), 8^(th), 9^(th) or 10^(th) round of sorting. Allclones retained wild-type Axl Ig1 sequence (not shown). **If cysteinesare present in loop, then total loop length and “shortened” loop lengthare noted. ⁺Contains in-frame G₃S (SEQ ID NO: 78) insertion in (G₄S)₃linker (SEQ ID NO: 79). #rpt: number of times that clone occurred in therandomly selected clones for sequencing.

Example 5 Characterization of Axl Variants to Gas6

In order to use yeast display to characterize the binding interactionsbetween Gas6 and the engineered EA mutants, we first sought to confirmthat yeast display allows accurate affinity measurements of the Gas6-Axlinteraction. Using yeast displayed Axl we were able to recapitulatepreviously reported binding affinities of Axl variants determined bysurface plasmon resonance and solid phase binding (Table 4). Thisvalidates that yeast-displayed Axl is similar to recombinant versions ofthe receptor.

TABLE 4 Comparison of affinity of Axl point mutants by yeast surfacedisplay (YSD) to values reported in the literature. K_(D) (nM) SolidYSD* phase ⁺ SPR ⁺ Wt Axl  1.7 ± 0.6 1 6 ± 2 E56R 10.2 ± 3.6 6 10 ± 2 E59R 109.2 ± 17.6 40 98 ± 24 T77R >200 >200 311 ± 118 *This work ⁺ Fromref (Sasaki et al., 2006 Structural basis for Gas6-Axl signalling, EMBOJ. 2006 Jan. 11; 25(1): 80-87.)

The affinities of the EETI-II mutants alone were too weak to bedetected, but when fused to Axl Ig1, the EA mutants exhibitedsubnanomolar affinities up to ˜4-fold stronger than wild-type Axl Ig1.Wild-type EETI-II fused to the Axl N-terminus exhibited the sameaffinity as wild-type Axl. This further demonstrates the fusionconstruct does not interfere with the native Axl-Gas6 interaction, andthat affinity improvement is due to the EETI-II loop mutants, ratherthan simply resulting from fusion of the EETI-II knottin to the AxlN-terminus (FIG. 4 and Table 4).

TABLE 5 Affinity of wt EETI-Axl and EA (EETI-II-axl fusion) mutants.x-fold K_(D) (nM) over wt Wt EETI-Axl 1.6 ± 0.3 1 EA 7.01 0.46 ± 0.063.6 EA 7.06 0.42 ± 0.11 3.9 EA 8.04 0.59 ± 0.08 2.8 Affinities arereported as avg. ± std. dev. of three independent experiments.

To explore the nature of the enhanced binding, we conducted bindingstudies to monitor dissociation kinetics. Incubation of yeast expressingeither wild-type Axl Ig1 or EA mutants with 2 nM Gas6 was followed byincubation with a molar excess of competitor in a similar manner to the‘off-rate’ sorts described above. While wild-type Axl Ig1 exhibitskinetic dissociation that is well-described by a single exponentialdecay model, the EA mutants exhibit more complex kinetics and must befit using a double exponential decay model (FIG. 5 and Table 5). As acontrol, wild-type EETI-Axl exhibited indistinguishable dissociationkinetics from wild-type Axl Ig1 and was well-fit by a single exponentialdecay model (data not shown).

TABLE 6 Kinetic dissociation constants of wild-type Axl Ig1 and EAmutants. k_(off, l) (hr) k_(off, 2) (hr) Wt Axl 0.76 ± 0.16 — EA 7.010.77 ± 0.16 0.038 ± 0.004 EA 7.06 0.74 ± 0.27 0.067 ± 0.010 EA 8.04 0.62± 0.14 0.048 ± 0.001

Kinetic constants are reported as avg.±std. dev. of three independentexperiments.

To interrogate the contributions from each of the engineered loops tothe enhanced affinity, we individually reverted loops 1 or 3 of the EAmutants to the wild-type EETI-II sequence and tested binding to Gas6(FIG. 6). In these studies wild-type EETI-Axl was used as a control for“reversion” of both loops to wild-type. Evaluation of persistent bindingof EA 7.06 revealed only loop 3 contributes to the interaction withGas6, as reversion of loop 1 to wild-type EETI-II sequence (EA 7.06wtL1) exhibits identical persistent binding to the parental EA 7.06mutant (FIG. 6B). For EA 7.01 and EA 8.04, reversion of loop 1 towild-type EETI-II sequence (EA 7.01 wtL1 and EA 8.04 wtL1) exhibitsweaker persistent binding than the respective parental mutants, butstronger than wild-type EETI-Axl. Reversion of loop 3 to wild-type in EA7.01 wtL3 and EA 8.04 wtL3 completely abolished improvement overwild-type EETI-Axl (FIGS. 6A&C). Together, this demonstrates that for EA7.01 and EA 8.04, loop 3 is the main contributor, but both engineeredloops are necessary for maximum enhancement of binding, and for EA 7.06loop 3 is the sole contributor.

Example 6 Knottin Fusions with Mutated Receptor Fragment (EETI-II-AxlIg1)

The following example describes the preparation of Axl Ig1 receptorfragments fused to mutated EETI-II knottins engineered to bindintegrins, namely knottins 2.5D and 2.5F. 2.5D and 2.5F are bothvariants of the Ecballium elaterium trypsin inhibitor-II (EETI-II)knottin. These knottins were engineered to specifically bind to theα_(v)β₃, α_(v)β₅ and α_(v)β₃, α_(v)β₅, α₅β₁ integrins, respectively. Toaccomplish this, loop 1 of EETI-II was replaced with a randomizedsequence containing the integrin recognition tripeptide motif, RGD.Yeast surface display and fluorescence activated cell sorting (FACS) wasthen used to select for clones with the desired binding properties.These integrins are clinically important cancer targets and Axl is areceptor tyrosine kinase that is an emerging target for cancer treatmentas well. Axl overexpression has been linked to invasive and metastaticphenotypes of a variety of cancers, suggesting that antagonizing theinteraction between Axl and its native ligand, Gas6, could be oftherapeutic value.

Axl S6-1 and S6-2 are engineered versions of Axl Ig1 that bind to Axl'snative ligand, Gas6, with higher affinity than wild-type. Usingerror-prone PCR, mutants were introduced into the wild-type Axl Ig1 geneand the resulting mutant DNA library was expressed on the surface ofyeast. Using FACS, clones with improved binding to Gas6 were isolated.Clones S6-1 and S6-2 display 20- and 12-fold improvements in equilibriumbinding over wild-type, respectively, with improvements largely comingfrom enhanced off-rates. In addition to binding Gas6 tighter, S6-1 has a13° C. improvement in melting temperature over wild-type representing asignificant enhancement in stability.

Table 7 below shows the various peptides (EETI-II) and the Axl mutantsused.

Protein/ Engineered SEQ Scaffold Target Portion ID NO: EETI-IIαvβ3, αvβ5 Loop 1: 59 mutant 2.5D CPQGRGDWAPTSC EETI-II αvβ3, αvβ5,Loop 1: 60 mutant 2.5F α5β1 CPRPRGDNPPLTC Axl Ig1* Gas6 None Axl S6-1*Gas6 G32S, D87G, V92A, G127R** Axl S6-2* Gas6 E26G, V79M, V92A, G127E***Axl Ig1 consists of the first Ig domain, encompassing amino acids19-132 of full-length Ax1 (Genbank Accession NO. P30530) **Locations ofthese mutations are further indicated for clarity by bolding andunderlining in the sequences immediately below.

Amino Acid Sequences:

The amino acid sequences of wild-type EETI-II, 2.5D and 2.5F are givenabove. Single amino acid mutations and a deletion were introduced intothe Axl Ig1 receptor fragment as shown below, where bracketed [Ap] isomitted in EA fusions shown in Table 3:

SEQ ID NO: 61 Axl Ig1: [AP]RGTQA E ESPFV GNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDG QILELADSTQTQ V PLGEDEQ D DWIV VSQLRITSLQLSDTGQYQCLVFLGH QTFVSQPGYV G LEGLP SEQ ID NO: 62 Axl S6-1:[AP]RGTQAEESPFV S NPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQ G DWIV A SQLRITSLQLSDTGQYQCLVFLGH QTFVSQPGYV RLEGLP SEQ ID NO: 63 Axl S6-2: [AP]RGTQA GESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDG QILELADSTQTQ M PLGEDEQDDWIV ASQLRITSLQLSDTGQYQCLVFLGH QTFVSQPGYV E LEGLP

Fusion Construction:

Using standard cloning techniques, the genes encoding for the EETI-IImutant and Axl Ig1 were assembled into a single genetic construct codingfor the fusion protein. The EETI-II domain was fused to the N-terminusof Axl Ig1, resulting a fusion protein consisting of an N-terminalknottin domain followed by the Axl Ig1 domain. To improve the overallflexibility of the fusion, the final proline of EETI-II and the initialalanine and proline of Axl were removed. The DNA encoding for the fusionprotein was then ligated into both yeast expression and secretionplasmids. This fusion protein has been expressed on the surface of yeastto allow for binding studies, as well as produced solubly.

Data:

Briefly, yeast displayed 2.5D-Axl was used to test whether this fusionwas functional. The fusion's ability to binding to soluble α_(v)β₃integrin and Gas6 was measured and compared to binding levels seen in2.5D and Axl alone. The fusion displayed α_(v)β₃ binding affinities thatmatched that of 2.5D, while it maintained wild-type Axl's affinity forGas6, validating the fusion construct. Additionally, binding of eachsoluble target was tested in the presence of a saturating amount of thesecond target to test the fusion's ability to concurrently bind bothα_(v)β₃ integrin and Gas6. These binding levels were the same as whenthey were measured individually, suggesting that the fusion can indeedsimultaneously bind to both of its targets. Finally, to confirm that thefusion is stable, it was produced solubly in the yeast Pichia pastoris.Purified recombinant yields were on the order of 50-75 mg per liter.These proteins were tested for their ability to bind to cellstransfected to overexpress the α_(v)β₃ integrin. They displayedequilibrium binding consistent with that previously determined for 2.5D,further validating that fusing the two protein domains did notnegatively affect binding properties.

Fusion Function:

This knottin-Axl fusion will function as a multispecific moleculecapable of concurrently antagonizing both integrin binding as well asthe native Gas6/Axl interactions. Gas6 is a soluble ligand whereas theintegrins are cell surface receptors, allowing both targets to be boundat the same time. Binding of Gas6 will sequester the soluble ligand,preventing it from associating with, and subsequently activatingendogenous Axl receptor. Binding to integrin receptors will prevent themfrom binding to extracellular matrix proteins.

Example 7 Knottin Fusions to Improve Yields of Engineered Knottins

As described above, knottins can be difficult to produce recombinantly.By fusing them to a well-expressing protein, they can be expressed inhigh yields. Cleavage of the knottin can be accomplished by theinclusion of a protease site between the protein domains.

Fusion Construction:

Using standard cloning techniques, the genes encoding for the EETI-IImutant and Axl Ig1 were assembled into a single genetic construct codingfor the fusion protein. Both N and C-terminal knottin fusions werecreated, with the Tobacco Etch Virus (TEV) recognition site, ENLYFQG(SEQ ID NO: 80), being inserted between the protein domains. The genewas then ligated into a yeast expression plasmid and transformed intothe yeast Pichia pastoris.

Amino Acid Sequence:

underlined—EETI mutant (2.5D)bolded—TEV recognition siteitalics—Axl Ig1

SEQ ID NO: 64 N-terminal fusion: GCPQGRGDWAPTSCKQDSDCLAGCVCGPNGFCGSENLYFQG RGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFV SQPGYVGLEGLPThe EETI portion is underlined. The TEV recognition site is in bold.

SEQ ID NO: 65 C-terminal fusion:APRGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLP ENLYFQG GCPQGRGDWAPTSCKQDSDCLAGCVCGPNGFCGS

Both N and C-terminal fusions were produced with purified yields of ˜50mg per liter. The purified fusions were then subjected to proteolyticcleavage by TEV, which released the knottin domains. The knottins werethen further purified by FPLC to separate them from their fusionpartner. It should be noted that folded, functional EETI mutant 2.5Dcould not be expressed in yeast without the assistance of this fusionprotein.

It can be seen that the N-terminal fusion contains a linking sequencethat is in bold. In addition, a direct fusions was made without thelinking sequence, i.e. wherein the caroxy terminal serine of the 2.5DEETI/integrin peptide is fused directly to the arginine of the Axl Ig1domain. By fusing EETI 2.5D to Axl Ig1, a multi-specific molecule wasformed, capable of binding αvβ3/αvβ5 integrins and Gas6. Analysis of thecrystal structure of Axl suggested that the N-terminus was far enoughaway from secondary structural elements that a direct fusion to theknottin would be appropriate results using the direct fusion aredescribed in Example 9.

Example 8 AgRP Knottin Against α_(v)β₃ Integrin Fused to an EngineeredFragment of HGF (NK1) that Binds the Met Receptor

A dual-specific fusion protein was constructed by linking the AgRPmutant, 7A, with one of the tightest binding NK1 fragments, named Aras4.Aras4 is linked at the C-terminus of AgRP7A and there is no amino acidlinker between two domains.

The binding towards soluble α_(v)β₃ integrin and Met receptor wasmeasured using yeast surface display. The binding against 0.5 nM and 5nM of α_(v)β₃ integrin and Met was measured and compared with AgRP 7Aand Aras4 alone (FIG. 7). The bar graphs in FIG. 7 show that the fusionproteins have comparable binding affinities with the AgRP and NK1mutants towards α_(v)β₃ integrin and Met receptors, respectively. Thisindicates that the fusion protein can be expressed and their individualcomponents bind to their respective targets without steric interference.

The open reading frame of the fusion protein, AgRP7A-Aras4, wasincorporated into the pPICK9K plasmid and transformed into Pichiapastoris. The fusion protein was expressed in yeast culture according tothe manufacturer's instructions (Invitrogen), then purified by metalchelating chromatography through the hexahistidine tag (SEQ ID NO: 77).The scheme of the gene of this fusion protein is show in the box below.The protein sequence of the fusion protein, AgRP7A-Aras4 is listed inTable 8 and listed below.

Above is a scheme of the gene of the fusion protein in pPCI9K plasmid.SnaBI, AvrII and MluI are the restriction enzyme sites.

TABLE 8 The protein sequences of Knottin-NK1 Bolded: Flag-TagUnderlined: Knottins (AgRp7A, EETI2.5F) Italics: NK1 variants Name ofFusion the fusion Fusion Seq ID protein Knottin Partner Protein sequence1 (SEQ AgRP7A- The Agouti NK1 fragment DYKDDDDKPRGCVRLHESCLG ID Aras4related of HGF QQVPCCDPAATCYCSGRGDND NO(s): protein (Aras4) LVCYCRYAEGQGKRRNTIHEFKK 66 (AgRP) SAKTTLIKIDPALRIKTEKANTADQCANRCTRSKGLPFTCKAFVFDKA RKRCLWFPFNSMSSGVKKEFGHE FDLYENKAYIRDCIIGRGRNYRGTVSITKSGIKCQPWSAMIPHEHSFL PSSYRGEDLRENYCRNPRGEEGGPWCYTSDPEVRYEVCDIPQCSEVE TRHHHHHH 2 (SEQ AgRP7A- The Agouti NK1 fragmentDYKDDDDKPRGCVRLHESCLG ID NO: M2.2 related of HGF QQVPCCDPAATCYCSGRGDND85): protein (M2.2) LVCYCRYAEGQRKRRNTIHEFKK (AgRP)SAKTTLIKIDPALKIKTEKVNTADQ CANRCTRNKG LPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYENKDYI RDCIIGNGRSYRGTVSITKSGIKCQ PWSSMIPHEHSFLPSSYRGEDLRENYCRNPR GEEGGPWCFTSDPEVRYEVCDIP QCSEVETRHHHHHH 3 (SEQAgRp7A- The Agouti NK1 fragment DYKDDDDKPRGCVRLHESCLG ID NO: M2.2related of HGF QQVPCCDPAATCYCSGRGDND 86) (D127A) protein (M2.2 LVCYCRYAEGQRKRRNTIHEFKK (AgRP) (D127A)) SAKTTLIKIDPALKIKTEKVNTADQCANRCTRNKGLPFTCKAFVFDKA RKRCLWFPFNSMSSGVKKEFGHE FDLYENKDYIRACIIGNGRSYRGTVSITKSGIKCQPWSSMIP HEHSFLPSSYRGEDLRENYCRNPR GE EGGPWCFTSDPEVRYEVCDIPQCSEVETRHHHHHH 4 (SEQ EETI2.5F- Ecballium NK1 fragmentDYKDDDDKPRGCPRPRGDNPP ID NO: Aras4 elaterium of HGFLTCSQDSDCLAGCVCGPNGFCG 87): trypsin (Aras4) YAEGQGKRRNTIHEFKKSAKTTLIinhibitor KIDPALRIKTEKANTADQCANRCT (EETI) RSKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYEN KAYIRDCIIGRGRNYRGTVSITKSGIKCQPWSAMIPHEHSFLPSSYRGE DLRENYCRNPRGEEGGPWCYTSD PEVRYEVCDIPQCSEVETRHHHHHH 5 (SEQ EETI2.5F- Ecballium NK1 fragment DYKDDDDKPRGCPRPRGDNPP ID NO:M2.2 elaterium of HGF LTCSQDSDCLAGCVCGPNGFCG 88): trypsin (M2.2)YAEGQRKRRNTIHEFKKSAKTTLI inhibitor KIDPALKIKTEKVNTADQCANRCT (EETI)RNKGLPFTCKAFVFDKARKRCLW FPFNSMSSGVKKEFGHEFDLYENKDYIRDCIIGNGRSYRGTVSITKSG IKCQPWSSMIPHEHSFLPSSYRGEDLRENYCRNPRGEEGGPWCFTSD PEVRYEVCDIPQCSEVETRHHHH HH 6 (SEQ EETI2.5F-Ecballium NK1 fragment DYKDDDDKPRGCPRPRGDNPP ID M2.2 elaterium of HGFLTCSQDSDCLAGCVCGPNGFCG NO: 89) (D127A) trypsin (M2.2YAEGQRKRRNTIHEFKKSAKTTLI inhibitor (D127A)) KIDPALKIKTEKVNTADQCANRCT(EETI) RNKGLPFTCKAFVFDKARKRCLW FPFNSMSSGVKKEFGHEFDLYENKDYIRACIIGNGRSYRGTVSITKSGI KCQPWSSMIPHEHSFLPSSYRGEDLRENYCRNPRGEEGGPWCFTSD PEVRYEVCDIPQCSEVETRHHHH HH

Variant sequences of the NK1 fragment could be used, and are described,e.g., in Hartman et al., “A functional domain in the heavy chain ofscatter factor/hepatocyte growth factor binds the c-Met receptor andinduces cell dissociation but not mitogenesis,” Proc. Nat. Acad. Sci.USA Vol. 89, pp. 11574-11578, December 1992.

The detail of the protein above (SEQ ID NO: 66) is shown below:

 Flag-Tag                   AgRP7A (between slashes)DYKDDDDKPR//GCVRLHESCLGQQVPCCDPAATCYCSGRGDNDLVCYCR//YAEG              Loop 1 Loop 2 Loop 3   Loop 4                                NK1QGKRRNTIHEFKKSAKTTLIKIDPALRIKTEKANTADQCANRCTRSKGLPFTCKAFVFDKARKRCLWFPFNSMSSGVKKEFGHEFDLYENKAYIRDCIIGRGRNYRGTVSITKSGIKCQPWSAMIPHEHSFLPSSYRGEDLRENYCRNPRGEEGGPWCYTSDPEVRYEVCDIPQC SEVETR HHHHHH

The His tag is underlined at the C terminus. The binding affinity of theAgRP7A-Aras4 fusion protein was measured on K562-α_(v)β₃ cells, whichexpress both α_(v)β₃ integrin and Met-receptor (FIG. 8). K562 leukemiacells were previously transfected with α_(v)β₃ integrin (Blystone, S. D.(1994). J. Cell Biol. 127, 1129-1137). We also showed by flow cytometrythat these cell lines also naturally express Met receptor (data notshown).

Knottins (EETI2.5F and AgRp7A) and NK1 fusion proteins were created andpurified for the study of in vitro biological characteristics. Threedifferent NK1 variants were fused to C-terminus of the two distinctknottin proteins, including M2.2, M2.2(D127A) and Aras4. Therefore, sixproteins composed of the following variations: AgRp7A-Aras4,EETI2.5F-Aras4, AgRp7A-M2.2, EETI2.5F-M2.2, AgRp7A-M2.2(D127A) andEETI2.5F-M2.2(D127A) were constructed and used for the in vitro assays.M2.2 was from the second round of directed evolution, Aras4 was from thethird round of directed evolution from our previous NK1 filing. D127A isa point mutant that has previously been shown to modulate antagonisticactivity).

In K562-α_(v)β₃ cell binding assays, the binding affinities (K_(D)values) of AgRp7A-M2.2(D127A) and EETI2.5F-M2.2(D127A) towards theα_(v)β₃ integrin in K562 cells transfected to express this integrin are2.1±1.1 nM and 4.6±1.6 nM. In HUVEC cell binding assays, the bindingaffinities (K_(D) values) of AgRp7A-M2.2(D127A) and EETI2.5F-M2.2(D127A)towards human umbilical vein endothelial cells (HUVECs) are 9.4±1.0 nMand 4.7±0.6 nM. HUVECs express medium levels of the α_(v)β₃, α_(v)β₅integrins, the Met receptor, and a high level of the α₅β₁ integrin.

In addition, a dual receptor direct binding assay showed thatmulti-specific proteins bind to Met and integrins simultaneously. Inthis experiment, a mixture of soluble Alexa-488 labeled human Met-Fc(220 nM) and the mono-specific and the multi-specific proteins (2 or 20nM) were added to K562-αvβ3 cells. Binding was detected by flowcytometry. AgRp7A-M2.2, EETI2.5F-M2.2, AgRp7A-M2.2(D127A) andEETI2.5F-M2.2(D127A) were able to bind to soluble Met-Fc while engagedwith α_(v)β₃ integrin on K562-α_(v)β₃ cells. These results demonstratethat the knottin fusions can simultaneously bind to α_(v)β₃ integrin andMet receptor.

Serum stability of AgRp7A-M2.2(D127A) and EETI2.5F-M2.2(D127A) was shownwhen the proteins were incubated with 40% human serum at 37° C. for overseveral days. Samples were analyzed by Western Blot and detected with anantibody against the FLAG epitope tag. No significant decrease in theamount of intact fusion protein was observed over 7 days, indicatingstability of the knottin fusion proteins to serum proteases and elevatedtemperatures.

A HUVEC proliferation assay was performed where cells were stimulatedwith 0.5 nM HGF. AgRp7A, EETI2.5F, AgRp7A-M2.2(D127A), orEETI2.5F-M2.2(D127A) proteins were added to observe their effects on theinhibition of HUVEC proliferation. AgRp7A had little inhibitory effecton HUVECs proliferation. EETI2.5F alone showed good inhibition (70%inhibition at 1 μM, where cells alone=90% inhibition). The knottinfusion proteins AgRp7A-M2.2(D127A) and EETI2.5F-M2.2(D127A) showedhigher inhibitory effects on cell proliferation compared AgRp7A andEETI2.5F, approaching inhibition levels equivalent to that of thenegative control.

A Met receptor phosphorylation assay was performed in PC3 (prostatecancer cells). Met receptor phosphorylation was assayed by Western blotafter stimulation with 0.3 nM HGF. AgRp7A, Aras4 and AgRp7A-Aras4proteins were added to observe their effects on the inhibition of Metphosphorylation. AgRp7A did not show inhibition of Met phosphorylation.Dose dependent decreases in Met receptor phosphorylation were observedupon addition of Aras4 and AgRP7A-Aras4, with slightly higher inhibitoryeffects observed with the AgRP7A-Aras4 knottin fusion protein. (Note:PC3 cells express medium levels of the α_(v)β₃ integrin and Met, and lowlevels of the α₅β₁ integrin).

Inhibition of PC3 cell adhesion to vitronectin was performed by coatinghuman vitronectin onto the wells of a microtiter plate and seeding cellsin the presence of varying concentrations of Aras4, AgRp7A, EETI2.5F,AgRp7A-Aras4, or EETI2.5F-Aras4. Half-maximal inhibitor concentrationvalues for all constructs were similar and in the low nM range (˜20-40nM), except for Aras4, which did not inhibit PC3 cell adhesion, asexpected.

Example 9 Knottin Fusion Directly Fused to Wild Type Axl ReceptorFragment

As described in Example 6, a direct fusion of the EETI knottin/integrinbinding peptide to an Axl membrane bound kinase receptor was prepared.The Axl Ig1 domain, amino acids 21-132 was used. By fusing EETI 2.5D toAxl Ig1, a multi-specific molecule was formed, capable of bindingαvβ3/αvβ5 integrins and Gas6.

The sequence is given below, where the knottin portion, 2.5D isunderlined, and the Axl portion begins with the sequence RGT . . . .

(SEQ ID NO: 84) GCPQGRGDWAPTSCSQDSDCLAGCVCGPNGFCGS/RGTQAEESPFVGNPGNITGARGLTGTLRCQLQVQGEPPEVHWLRDGQILELADSTQTQVPLGEDEQDDWIVVSQLRITSLQLSDTGQYQCLVFLGHQTFVSQPGYVGLEGLP.The ability of the fusion protein to bind to either αvβ3 integrin orGas6 was tested using the yeast display platform, wherein the EETI2.5D-Axl fusion protein was cloned into a yeast display construct anddisplayed on the cell surface. Yeast expressing either EETI 2.5D, AxlIg1, or the EETI 2.5D-Axl fusion protein were incubated with varyingconcentrations of soluble αvβ3 integrin or Gas6. The binding reactionswere allowed to come to equilibrium at which time excess ligand wasremoved by washing. Yeast were resuspended in a solution containingfluorescently labeled antibodies against the appropriate ligand(integrin or Gas6). Flow cytometry was used to quantify bound integrinor Gas6 through the detection of the secondary antibodies. Theseexperiments, showed that EETI 2.5D and Axl Ig1 only bind αvβ3 integrinand Gas6, respectively, whereas the EETI 2.5D-Axl fusion binds bothproteins at levels equivalent to their mono-specific components. Thisdata demonstrates that the fusion of EETI 2.5D and Axl Ig1 does notdisrupt binding to either target protein. Yeast expressing EETI 2.5D, wtAxl Ig1 or EETI 2.5D-Axl fusion were incubated with 20, 50 or 100 nMαvβ3 integrin. As expected, only EETI 2.5D and EETI 2.5D-Axl bind tointegrin, as wt Axl has no native affinity towards this receptor. Thesame set of yeast samples were incubated with 0.2, 2 or 20 nM Gas6.Wild-type Axl and EETI 2.5D-Axl show affinity for Gas6, whereas nobinding is detected to EETI 2.5D alone. In both cases, the EETI 2.5D-Axlfusion protein binds to integrin or Gas6 with affinities similar to itscorresponding mono-specific components.

Next, the ability of the fusion to bind to both targets simultaneouslywas investigated by incubating yeast expressing EETI 2.5D-Axl with αvβ3integrin in the presence of a saturating concentration of Gas6, or withGas6 in the presence of a saturating concentration of αvβ3 integrin.These results are outlined in FIG. 9. In both cases, the presence of anexcess of the soluble second ligand does not substantially diminishbinding to the primary ligand. These results indicate that binding ofone target to the EETI 2.5D-Axl fusion protein does not prevent bindingof the second, permitting simultaneous interactions with both Gas6 andαvβ3 integrin.

Referring to FIG. 9, yeast-surface display binding data. In FIG. 9A,yeast were incubated with 20 or 100 nM Gas6 in the presence of 200 nMαvβ3 integrin. The bispecific protein maintains affinity to Gas6 whenexcess integrin is present. In FIG. 9B, yeast were incubated with 100 or200 nM αvβ3 integrin in the presence of 100 nM Gas6. Affinity to αvβ3integrin is not lost when Gas6 is present. Together, these experimentssuggest that EETI 2.5D-Axl is capable of simultaneously binding to bothtargets.

Example 10 Knottin Fusion Produced in Recombinant Yeast

The EETI 2.5D-Axl fusion protein was then cloned into the pPic9K yeastsecretion vector and soluble protein was recombinantly produced in theyeast strain P. pastoris according to the manufacturer's manual(Invitrogen). Protein was purified from culture supernatant using nickelaffinity chromatography and heterogeneous yeast glycosylations werecleaved by treating the protein with endoglycosidase (endoH). MonomericEETI 2.5D-Axl protein was further purified using size exclusionchromatography. The purity of the final product was analyzed usingSDS-PAGE, and analytical size exclusion chromatography. Highly pure,monomeric EETI 2.5D-Axl fusion protein was obtained at an approximateyield of 35 milligrams per liter.

Recombinantly produced EETI 2.5D-Axl was tested for its ability to bindcell-surface αvβ3 integrin. K562 leukemia cells that have beentransfected to overexpress αvβ3 integrin (K562-αvβ3 cells) wereincubated with varying concentrations of EETI 2.5D-Axl. Once thereactions reached equilibrium, excess EETI 2.5D-Axl was removed bywashing and cells were resuspended in a solution containing afluorescently labeled antibody against the FLAG epitope tag on therecombinant multispecific protein. Flow cytometry was then used toquantify the amount of bound EETI 2.5D-Axl by detecting the fluorescentanti-FLAG antibody. The affinity (Kd) of the EETI 2.5D-Axl fusionprotein to the K562-αvβ3 cells was determined to be 1.72 nM.Additionally, circular dichroism spectroscopy was used to analyze thethermal stability of the EETI 2.5D-Axl fusion protein as compared to wtAxl. Wild-type Axl Ig1 was found to have a melting temperature (Tm) of41° C. By fusing EETI 2.5D to the N-terminus of Axl, an improvement of11° C. in stability was observed (Tm˜52° C.). The results of thesebinding studies and CD experiments are summarized in the table below.

K_(D) to αvβ3 Tm (° C.) integrin (nM) wt Axl Ig1 41 ± 0.6 — 2.5D - AxlIg1 52 ± 0.7 1.72

The specificity of the observed binding to the αvβ3 integrin expressedon K562-αvβ3 cells was tested by incubating the cells with EETI 2.5D-Axland cyclic RGD (cRGD). As EETI 2.5D binds to the same epitope on theintegrin as the cRGD, a molar excess of cRGD will compete off EETI2.5D-Axl if the protein is binding specifically to the integrins. cRGDinhibits the binding of EETI 2.5D-Axl to K562-αvβ3 cells suggesting theprotein is indeed binding specifically to αvβ3 integrin on the cellsurface.

Example 11 Self-Cleaving TEV-Knottin Fusion

Several knottins are difficult to produce recombinantly as they producehigh order oligomers rather than properly folded monomers. For example,while we have demonstrated robust methods for chemical synthesis and invitro folding of EETI 2.5D using solid phase peptide synthesis, we havenot been able to recombinantly express this knottin in a yeast-basedexpression system. The observation that properly folded EETI 2.5D-Axlfusions could be produced in high recombinant yield led to thedevelopment of a self-cleaving TEV-2.5D construct as a means torecombinantly produce knottins.

In this fusion, the Tobacco Etch Virus (TEV) protease was fused to theEETI knottin variant 2.5D. TEV recognizes the eight amino acid sequence,which can be either SENLYFQS or GENLYFQG (SEQ ID NO: 82) wherein glycine(G) may be substituted with serine (S) in the amino acid sequence. TEVthen cuts just prior to the last G/S. This cleavage site was placed atthe C-terminus of the TEV protease, followed by EETI 2.5D. The firstamino acid in EETI 2.5D is a glycine (G), thus to eliminate extraresidues from being left post-cleavage, that glycine was removed. Upontranslation, the protease portion of the fusion protein can interactwith the cleavage sites of another fusion, cutting it and therebygenerating free EETI 2.5D knottin.

This autocleaving fusion protein was cloned into the pPic9K yeastsecretion vector with N and C-terminal FLAG and 6×HIS tags,respectively, and transformed into the yeast strain P. pastorisaccording to the manufacturer's directions (Invitrogen). Western blotson the supernatant of expression cultures were probed for either theFLAG or HIS tag. The blots revealed that probing for the N-terminal flagtag shows a high molecular weight species corresponding to the TEVprotease. Blots stained for the C-terminal 6×HIS tag show a ˜8 kDaspecies which corresponds to the cleaved knottin. Based on theseexpression tests, this autocleaving construct is a viable method torecombinantly express knottins which are difficult to produce instandard microbial systems.

The autocleaving construct permits recombinant production of knottinsotherwise incapable of being produced in microbial systems.—Thisstrategy could also be used to produce proteins besides knottins.Alternatively, a fusion partner such as Axl could be used to facilitaterecombinant expression of knottins, with a protease cleavage siteintroduced in between the knottin and Axl proteins.

Example 12 Knottin-Fc Fusions

In this example, a mouse antibody Fc portion is fused to an integrinbinding knottin, EETI as described above. Knottin-Fc fusions werecreated by molecular cloning and mammalian cell expression. Thesemodified knottin proteins will have long circulation times (days)compared to unmodified knottins, which have half-lives on the order ofminutes. Using this system, we showed that EETI-based knottin peptides2.5D and 2.5F, and wild-type EETI-II, can be fused to an Fc domain ofmouse Ig2a (SEQ ID NO: 83), and recombinantly expressed knottin-Fcfusion proteins in mammalian human embryonic kidney (HEK) cells. The Fcdomain is a known sequence, see, e.g. Accession NM_(—)010184.2 for anmRNA sequence. The knottin peptides were purified and run on a NuPAGE4-12% Bis-Tris gel. The results showed the expected sizes of non-reduced(NR) and reduced (R) knottin 2.5D-Fc. The knottin proteins were thenanalyzed by gel filtration chromatography in which the purifiedknottin-Fc protein 2.5D showed no tendency to aggregate.

The binding of the knottin-Fc proteins to tumor cell lines were thenmeasured. The knottin 2.5F-Fc peptide was found to bind with a greateraffinity to sk0v3 cells compared to the knottin 2.5D-Fc peptide whenmeasured against wild-type EETII-Fc. In contrast, knottin 2.5-Fc and2.5D-Fc bound with similar affinity to K562 leukemia cells transfectedwith αvβ3 integrin.

In another tumor model, the ability of the knottin-Fc proteins toinhibit PC3 cell adhesion to the extracellular matrix (ECM) proteinvitronectin was analyzed. Both of the knottin-Fc proteins stronglyinhibited tumor cell adhesion, while the negative control did not.Results are shown in FIG. 10. As the inhibition of integrin-ECM adhesioninduces caspase-mediated apoposis, this biological mechanism will beexplored in future studies.

This work is the first demonstration that an antibody Fc domain can befused to a knottin protein without disrupting receptor binding affinity.This strategy will be a general platform for increasing half-life ofengineered knottin proteins against a variety of biomedical targetsbesides integrins. It is also a potential platform to make dimericproteins (as Fc fusions are bivalent), which can have increased bindingaffinity and increased or altered biological potency over monovalentknottins. Furthermore, Fc fusions can be used as a framework toconstruct higher order oligomers or multivalent/multispecific proteins,similar to what has been done with antibody-based agents.

CONCLUSION

The above specific description is meant to exemplify and illustrate theinvention and should not be seen as limiting the scope of the invention,which is defined by the literal and equivalent scope of the appendedclaims. Any patents or publications mentioned in this specification areintended to convey details of methods and materials useful in carryingout certain aspects of the invention which may not be explicitly set outbut which would be understood by workers in the field. Such patents orpublications are hereby incorporated by reference to the same extent asif each was specifically and individually incorporated by reference andcontained herein, as needed for the purpose of describing and enablingthe method or material referred to.

1. A fusion protein, comprising: (a) a knottin polypeptide havingtherein a knottin scaffold comprising therein a binding loop having anon-native sequence for binding to a first target; and (b) a secondpolypeptide having therein a sequence for binding to a second target,said second polypeptide being either (i) a cell surface receptor bindingto said second target or (ii) a cell surface receptor ligand. binding tosaid second target.
 2. The fusion protein of claim 1 wherein the knottinpolypeptide is one of EETI-II, AgRP, or agatoxin.
 3. The fusion proteinof claim 1 wherein the knottin polypeptide is ω-conotoxin.
 4. (canceled)5. The fusion protein of claim 1 wherein the non-native sequencemediates attachment between a cell and the tissues surrounding it. 6.The fusion protein of claim 5 wherein the knottin polypeptide contains asequence that mediates binding to one or more of (a) alpha v beta 3integrin, (b) alpha v beta 5 integrin, and (c) alpha 5 beta 1 integrin.7. The fusion protein of claim 1 wherein the second polypeptide is anextracellular domain of a receptor tyrosine kinase.
 8. The fusionprotein of claim 7 wherein the second polypeptide is a receptor tyrosinekinase Ig1 domain.
 9. The fusion protein of claim 7 wherein the Ig1domain is from Axl, MuSK, or the FGF receptor.
 10. The fusion protein ofclaim 8 wherein the receptor tyrosine kinase is an Axl receptor.
 11. Thefusion protein of claim 9 wherein the knottin polypeptide is selectedfrom the group consisting of EETI-II, AgRP, and agatoxin.
 12. The fusionprotein of claim 11 wherein the binding loop is engineered to bind toone or more of (a) alpha v beta 3 integrin, (b) alpha v beta 5 integrin,and (c) alpha 5 beta 1 integrin.
 13. A fusion protein according to claim1, comprising: (a) an EETI-II or AgRP knottin polypeptide comprising abinding loop with high affinity to an integrin; and (b) a polypeptideselected from the group consisting of (i) an Axl extracellular domainand (ii) NK1 fragment of hepatocyte growth factor.
 14. A method forpreparing a fusion protein according to claim 1, comprising the stepsof: (a) preparing a library having a number of DNA constructs encodingthe fusion protein and a number of randomized DNA sequences within theDNA constructs; (b) expressing the DNA constructs in the library inyeast, wherein expressed DNA constructs are displayed as polypeptideswith randomized sequences on the yeast surface; (c) screening the clonesfor binding of the expressed DNA constructs to the first target or thesecond target by contacting the clones with a target; (d) selectingclones that express translated DNA constructs that bind with highaffinity to the target; and (e) obtaining the coding sequences of theselected clones, whereby said fusion protein may be prepared.
 15. Themethod of claim 14 wherein the knottin is one of EETI-II, AgRp,agatoxin, or conotoxin.
 16. The method of claim 15 wherein the secondpolypeptide is selected from the group consisting of a receptor fragmentcomprising a ligand binding site and a ligand fragment comprising a sitethat binds to a receptor for the ligand.
 17. The method of claim 15wherein the second polypeptide is a tyrosine kinase receptor fragment.18. The method of claim 16 wherein the second polypeptide is a growthfactor fragment.
 19. The method of claim 16 wherein the secondpolypeptide comprises a polypeptide selected from the group consistingof Axl, c-Met, HGF, VEGF, VEGF receptor, and Gas6.
 20. The method ofclaim 16 wherein the knottin polypeptide is EETI-II.
 21. The method ofclaim 15 wherein the knottin is engineered to bind to an integrin. 22.The method of claim 21 wherein the integrin is at least one of (a) alphav beta 3 integrin, (b) alpha v beta 5 integrin, and (c) alpha 5 beta 1integrin.
 23. The method of claim 15 wherein the knottin is EETI-IIengineered in loop 1 and loop
 3. 24. A method for inhibiting binding ofa ligand to a receptor, comprising the steps of: (a) administering anamount of a soluble fusion protein comprising (i) a polypeptide encodingan extracellular domain of a receptor to be inhibited and (ii) a knottinpolypeptide having a loop domain engineered to bind to a cell surfacereceptor that is not the receptor to be inhibited.
 25. The method ofclaim 22 wherein the cell surface receptor is an integrin.
 26. Themethod of claim 22 wherein the receptor to be inhibited is a receptortyrosine kinase.
 27. The method of claim 25 wherein the tyrosine kinaseis a TAM receptor tyrosine kinase.
 28. A fusion protein according toclaim 1 having at least 90% sequence identity to a sequence that is oneof (a) a fusion of AgRP and NK1, as set forth in SEQ ID NO: 66, SEQ IDNO: 85, or SEQ ID NO: 86; or (b) a fusion of EETI and NK1, as set forthin SEQ ID NO: 87, SEQ ID NO: 88, or SEQ ID NO:
 89. 29. A fusion proteinaccording to claim 1 having at least 90% sequence identity to anEETI-Axl1 fusion as set forth in SEQ ID NO: 84.